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Vol. 17, Issue 12, 5004-5016, December 2006

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The Formin mDia Regulates GSK3 through Novel PKCs to Promote Microtubule Stabilization but Not MTOC Reorientation in Migrating Fibroblasts

Christina H. Eng^*,, Thomas M. Huckaba^*, and Gregg G. Gundersen^*

*Department of Anatomy and Cell Biology and Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University, New York, NY 10032

Submitted October 3, 2005; Revised August 14, 2006; Accepted September 6, 2006
Monitoring Editor: Erika Holzbaur

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3 (GSK3) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3 inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3 in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3 functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3 phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3 through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3 Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3 through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Microtubule (MT) arrays become polarized to perform their functions in development, differentiation, and cell migration. One of the major ways MT arrays become polarized is through the generation of a subset of stabilized MTs (Gundersen and Bulinski, 1988; Bulinski and Gundersen, 1991; Gundersen et al., 2004). In contrast to dynamic MTs, selectively stabilized MTs display longer half-lives and increased resistance to MT-depolymerizing drugs (Webster et al., 1987; Khawaja et al., 1988). Selectively stabilized MTs are abundant in differentiated cells, but are not restricted to differentiated cells: in wounded monolayers of fibroblasts, stable MTs form selectively so that they orient toward the leading edge (Gundersen and Bulinski, 1986; Gundersen and Bulinski, 1988; Palazzo et al., 2004). Selective MT stabilization is thought to occur by the formation of a plus-end cap that prevents the MT from growing or shrinking (Infante et al., 2000), and factors implicated in selective MT stabilization are localized at the ends of these MTs (Wen et al., 2004). Stabilized MTs accumulate posttranslationally modified tubulin, including detyrosinated and acetylated tubulin, and this may contribute to the recognition of stabilized MTs by factors that selectively interact with modified tubulin, such as kinesin motors (Liao and Gundersen, 1998). The functions of these stabilized, detyrosinated MTs in polarized cells are beginning to be understood, and studies have highlighted their importance in vimentin intermediate filament extension (Gurland and Gundersen, 1995; Kreitzer et al., 1999), endosomal recycling (Lin et al., 2002), migration (Wen et al., 2004), and virus infection (Naranatt et al., 2005). In addition, mice lacking the detyrosination/tyrosination cycle die shortly after birth and have neuronal defects (Erck et al., 2005).

In mammalian fibroblasts, the formation of long-lived stable MTs is regulated by the serum factor lysophosphatidic acid (LPA), which stimulates a signaling pathway that involves the heterotrimeric G protein, G_12/13, the small GTPase Rho, its effector, the formin mDia, and the MT plus-end binding proteins EB1 and APC (Cook et al., 1998; Palazzo et al., 2001a; Wen et al., 2004; Goulimari et al., 2005). Rho and mDia also appear to be involved in MT stabilization in embryonic endodermal cells, where they function along with the plakin, ACF-7 (Kodama et al., 2003). Binding of activated Rho to mDia is thought to relieve mDia's autoinhibited state and lead to its activation (Alberts, 2001). Activated forms of mDia bind both EB1 and APC, and all three proteins can be found on the ends of stable MTs, consistent with a capping mechanism (Wen et al., 2004). MTs stabilized by this Rho-mDia signaling pathway do not grow or shrink (Cook et al., 1998; Palazzo et al., 2001a; Wen et al., 2004), making this form of stabilization different from that of MAPs, which stabilize MTs by binding along their length, but do not block tubulin exchange at the ends.

The MT cytoskeleton is also polarized by positioning the MT organizing center (MTOC) at a specific place in the cell. In migrating fibroblasts, astrocytes, macrophages, and endothelial cells, the MTOC is reoriented so that it lies between the nucleus and the leading edge (Gotlieb et al., 1981; Kupfer et al., 1982, 1983; Gundersen and Bulinski, 1988; Etienne-Manneville and Hall, 2001). Cdc42, Par6, atypical PKC (aPKC), and dynein comprise a common "MTOC reorientation" pathway in fibroblasts, astrocytes, and endothelial cells (Etienne-Manneville and Hall, 2001, 2003; Palazzo et al., 2001b; Tzima et al., 2003; Gomes et al., 2005). In fibroblasts, this pathway, like that regulating MT stabilization, is triggered by LPA (Palazzo et al., 2001b). In fibroblasts, it is known that LPA triggers a second Cdc42-regulated pathway that functions to move the nucleus rearward, whereas the Par6-aPKC-dynein pathway maintains the MTOC at the cell centroid. The combined effects of the two pathways are necessary for MTOC reorientation (Gomes et al., 2005). The second Cdc42-regulated pathway involves the Cdc42 effector, MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase), myosin and actin retrograde flow, which is thought to drive the nucleus rearward (Gomes et al., 2005).

GSK3 was named for its role in regulating glycogen synthase, although it is now known to participate in many signaling pathways and cellular activities. GSK3 is usually constitutively active and most signaling pathways result in the inhibition of GSK3. For example, insulin stimulates protein kinase B (PKB) to phosphorylate Ser9 (pSer9) of GSK3 and this inhibits its activity (Cross et al., 1995). Insulin treatment of starved 3T3 fibroblasts does promote a small increase in the levels of stable MTs (Gundersen et al., 1994), suggesting that regulation of GSK3 activity may influence stable MT formation. In the Cdc42 pathway involved in MTOC reorientation in astrocytes, activation of atypical PKC also increases GSK3 pSer9 levels (Etienne-Manneville and Hall, 2003), but whether GSK3 participates in MTOC reorientation in fibroblasts or other systems that orient their MTOC has not been tested.

LPA is another cytokine that causes the inhibition of GSK3 by phosphorylation on Ser9, and although PKCs seem to be involved, the specific isoform(s) has not been identified (Fang et al., 2002). Because LPA stimulates both MT stabilization and MTOC reorientation in starved NIH 3T3 fibroblasts, we were interested in whether the inhibition of GSK3 was important for the effects of LPA on either or both of these MT-polarizing events. Given the evidence that GSK3 is involved in MTOC reorientation in astrocytes, we predicted that it would also be involved in both processes in fibroblasts. However, we find that GSK3 is not involved in MTOC reorientation in fibroblasts, but instead is involved in MT stabilization. Because LPA-induced MT stabilization in fibroblasts is regulated by a Rho-mDia pathway, this suggested that there might be a novel pathway involved in regulating GSK3 in response to LPA stimulation. Indeed, we find that GSK3 pSer9 is stimulated by both LPA and active forms of mDia1 or mDia2 and that mDia1 was both necessary and sufficient for enhancing GSK3 pSer9. mDia1 appears to regulate GSK3 phosphorylation through a pathway involving PKC, a novel PKC isoform, because mDia1 directly binds PKC, and PKC was necessary for both the LPA- and mDia-stimulated phosphorylation of GSK3 and for formation of stable MTs. These results describe a novel activity for a formin in regulating kinase activity and show that in fibroblasts, GSK3 contributes to the Rho-mDia-EB1-APC pathway that regulates MT stabilization.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials, Cell Culture, and Microinjection
All materials were purchased from Sigma (St. Louis, MO) unless otherwise indicated. NIH 3T3 cells were maintained as previously described (Cook et al., 1998; Kreitzer et al., 1999; Palazzo et al., 2001a). For microinjection, confluent NIH 3T3 cells were serum starved for 2 d, wounded, and microinjected as previously described (Cook et al., 1998; Kreitzer et al., 1999; Palazzo et al., 2001a). Plasmid DNA was microinjected in H-KCl (10 mM HEPES, pH 7.4, 140 mM KCl) at 100–200 µg/ml and allowed to express 2–4 h after microinjection. To induce stable MTs and MTOC reorientation, 5 µM LPA was added for 90–120 min. GSK3 inhibitors LiCl (20 mM) or SB216763 (Tocris, Ballwin, MO) were added to starved cells and incubated for 2 h. SB216763 was used at 2–10 µM with similar results.

DNA Plasmids
All HA-tagged PKC constructs were a gift from J. W. Soh (Soh et al., 1999), and flag-tagged dominant negative PKC was from C. Carpenter (Coghlan et al., 2000). Full-length HA-Rok was from L. Lim (Leung et al., 1996). mDia1 and mDia2 constructs were from A. Alberts (Alberts, 2001), except for pEGFP-C1-521-1040-mDia2, which was made by subcloning 521-1040-mDia2 from pEFm-EGFP (Wen et al., 2004) into pEGFP-C1 (Clontech, Palo Alto, CA). HA-tagged GSK3 was constructed by PCR amplification of pcDNA3-GSK3 (a gift from J. Kitajewski) with primers 5'-GGCGGCGTCGACAGAAGAGCCATCATG-3' and 3'-GCCTCCAACTCTACCTCTAGAGCCCCC-5' and cloned into pKHA₃ (a gift from D. Brautigen). To prepare GFP-GSK3, GSK3 was subcloned from pKHA₃ into pEGFP-C1 (Clontech). The GFP-GSK3 fusion was then subcloned into pCAGGS/ES (a gift from P. Scheiffle) downstream of the chicken -actin promoter. All constructs were verified by sequencing, and DNA was purified with Qiagen midiprep kits (Chatsworth, CA).

mDia1 siRNA
COS-7 cells were transfected with mDia1 siRNA in a six-well dish with 4 µl of 20 µM siRNA and 10 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 1 ml of growth media (DMEM with 10% FBS). The mDia1 siRNA sequence was 5'-GCUGGUCAGAGCCAUGGAU-3' (Arakawa et al., 2003) and the control GFP sequence was 5'-GGCUACGUCCAGGAGCGCACC-3'. Both siRNAs were from Dharmacon (Boulder, CO). After 24 h, the medium was changed to serum-free DMEM for another 24 h. Cells were lysed and analyzed for GSK3 phosphorylation. Similar knockdown results in COS-7 cells were obtained with a second mDia1 siRNA constructed using the Ambion Silencer siRNA construction kit (Austin, TX). This mDia1 sequence was 5'-AAGGUGAAGGAGGACCGCUUU-3'. The GAPDH siRNA used was the control provided with the kit. For microinjection of siRNA into NIH 3T3 fibroblasts, confluent monolayers were serum-starved and wounded. A few hours later, Ambion kit-synthesized siRNAs were diluted in H-KCl and microinjected at 10 µM with 1 mg/ml 500 kDa FITC-dextran (Molecular Probes, Eugene, OR) as an injection marker. After microinjection (48 h), cells were fixed for 5–10 min in methanol at –20°C and processed for immunofluorescence. To visualize individual siRNA-treated cells, the Ambion kit-synthesized siRNA was labeled with CX-rhodamine using the Mirrus Label IT siRNA Tracker kit (Boston, MA) according to the manufacturer's instructions. NIH 3T3 fibroblasts passaged at least three times in FBS were transfected with 2 µl Mirrus TKO Transfection reagent and 200 nM siRNA in 300 µl of growth media in a 24-well plate. After transfection (24 h), cells were replated onto coverslips and allowed to adhere another 24 h. Cells were then starved for 24 h, treated with LPA, and fixed and processed for immunofluorescence as above. To examine GSK3 pSer9 levels (see below) in siRNA treated cells, NIH 3T3 cells were transfected with siRNA to mDia1 or GAPDH (Invitrogen) with Lipofectamine Plus. Cells were split 24 h after transfection and transfected a second time 48 h after transfection. After the second transfection, cells were washed, serum-starved overnight, treated with LPA or insulin, and processed for immunofluorescence (see below).

Immunofluorescence Microscopy
Staining of fixed cells was performed as previously described (Gundersen et al., 1984). Glu MTs were detected with a polyclonal rabbit antibody (Ab) SG that specifically recognizes detyrosinated (Glu) tubulin (Gundersen et al., 1984; 1:400), and tyrosinated (Tyr) MTs were detected with the rat mAb YL1/2 (1:10, European Collection of Animal Cell Cultures, Salisbury, United Kingdom). The following Abs were used for detection of overexpressed or endogenous proteins; mouse mAb to HA (12CA5, 1:100, Roche, Indianapolis, IN), mouse mAb to GSK3 (0011-A, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), mouse mAb to mDia1 (clone 51, 1:100, BD Transduction, Lexington, KY), and mouse mAb to GAPDH (6C5, 1:1000, Ambion). For certain experiments, cells were also stained with mouse mAb to actin (C4D6, 1:200, a gift from J. Lessard, University of Cincinnati), -tubulin (3F3, 1:200), or -tubulin (GTU-88, 1:200, Sigma). For localizing F-actin, cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and stained with rhodamine-phalloidin (1:500, Molecular Probes). Digital images were acquired as previously described (Palazzo et al., 2001a).

For staining with GSK3 pSer9 Ab (1:100, Cell Signaling Technology, Beverly, MA), cells were fixed with 2% paraformaldehyde + 0.2% glutaraldehyde in PBS for 20 min and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were then blocked in 2% BSA in TBS and then stained as previously described, except primary antibody incubations were for 1 h. To quantify GSK3 pSer9 in individual cells, digital images were acquired with equivalent exposure times as previously described (Palazzo et al., 2001a). The average fluorescence intensity of the entire cell was measured using MetaMorph software (Universal Imaging, West Chester, PA) on a per cell basis after subtracting the background intensity (measured in an adjacent cell-free area of the coverslip). We attempted to correct the fluorescence measurements for nonspecific staining of the secondary antibody; however, these readings were too variable to give consistent results between the experiments and so were not corrected for.

Analysis of GSK3 Phosphorylation Levels by Western Blotting
COS-7 cells were transfected in growth medium (DMEM with 10% CS) with Lipofectamine 2000 according to the manufacturer's specifications. After transfection (6–24 h), cells were washed twice with serum-free medium (SFM; DMEM + 10 mM HEPES) and incubated in SFM for an addition 18–24 h. Cells were treated with 10 µM LPA or 10–100 µg/ml insulin for 5 min. To inhibit PKC, 10 µM bisindolylmaleimide I (Calbiochem, La Jolla, CA) was added 1 h before treatment with LPA or insulin. Cells were washed once with cold PBS and lysed in 1% Triton X-100, 150 mM NaCl, 20 mM Tris, pH 7.4 or 8.0, phosphatase inhibitors (5 mM -glycerophosphate, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate), and Protease Inhibitor Cocktail (Sigma). After 10 min extraction on ice, lysates were clarified by centrifugation (13,000 x g, 10 min, 4°C). Protein concentration was determined with a BCA protein assay and equal amounts of protein were run on SDS-PAGE, transferred to nitrocellulose and analyzed by Western blotting with rabbit Ab to GSK3 pSer9 (1:5000, Cell Signaling Technology), mouse mAb GSK3 (0011-A, 1:5000, Santa Cruz), mouse mAb to mDia1 (clone 51, 1:1000, BD Transduction), rat mAb to HA (3F10, 1:5000, Roche), and mouse mAb to vinculin (VIN-11–5, 1:5000, Sigma).

Binding Experiments
COS-7 cells were transfected with myc-GFP-GBD-mDia1 or myc-GFP as a control along with pHACE-PKC, pHACE-PKC, pHACE-PKC, or HA-ROK plasmids using Lipofectamine 2000 according to the manufacturer's protocol. After 24 h transfection, cells were washed once with cold PBS and lysed in RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.4, 150 mM NaCl, and phosphatase and protease inhibitors). After 10-min extraction on ice, lysates were clarified by centrifugation (13,000 x g, 10 min, 4°C). To each extract, rabbit Ab to full-length GFP (1:500, BD Transduction) and protein A-Sepharose beads were added, and lysates were mixed overnight at 4°C. Beads were washed in RIPA buffer, eluted with SDS sample buffer and loaded on gels. Expressed proteins were detected by Western blotting with mouse mAb to myc (9E10, 1:5000, Santa Cruz) and rat mAb to HA (3F10, 1:5000, Roche). For endogenous binding, 5 mg of precleared mouse brain extract was immunoprecipitated with 20 µg rabbit anti-PKC (Santa Cruz) or control rabbit IgG overnight at 4°C in lysis buffer containing 1% TX-100, 150 mM NaCl, 20 mM Tris, pH 7.4, and phosphatase and protease inhibitors. Protein G-Sepharose beads were then added and mixed for an additional 3 h. Beads were washed with lysis buffer, eluted with SDS sample buffer, and analyzed by SDS-PAGE followed by Western blotting. Antibodies used for blotting were mouse mAb to mDia1 (1:1000, BD Transduction) and rabbit Ab to PKC (1:1000, Santa Cruz). For direct in vitro binding, 20 µg GST-521-1040-mDia2 or an equal molar amount of GST was bound to glutathione-agarose beads for 1–2 h at 4°C. Then, 6 pmol recombinant purified human PKC or PKC (Calbiochem) was added in lysis buffer with or without lipids (100 µg/ml phosphatidylserine and 10 µg/ml diacylglycerol; Avanti, Aalabaster, AL) and mixed overnight at 4°C. Beads were washed with lysis buffer, and bound proteins were eluted with SDS sample buffer and run on gels. Bound proteins were detected by Western blotting with rabbit Ab to PKC (1:2000, Santa Cruz), rabbit Ab to PKC (1:2000, Upstate Biotechnology, Lake Placid, NY), or mouse mAb to GST (1:20,000, Cell Signaling Technology). For blot overlay analysis, equimolar amounts of PKC or PKC were subject to SDS-PAGE and transferred to nitrocellulose. The membrane was incubated overnight at 4°C with 10 µg/ml GST-521-1040-mDia2 or an equimolar amount of GST in TBST containing 5% BSA, 100 µg/ml phosphatidylserine and 10 µg/ml diacylglycerol. The bound GST-mDia2 or GST was detected by mouse Ab to GST, and quantified using the LI-COR Odyssey Infrared Imaging System (Cincinnati, OH).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
GSK3 Is a Negative Regulator of LPA-stimulated MT Stabilization
We first tested whether GSK3 was involved in the LPA-stimulation of MT stabilization and/or MTOC reorientation in wound-edge serum-starved NIH 3T3 fibroblasts. For MT stabilization, we assayed the formation of stabilized MTs by immunofluorescently staining cells for post-translationally detyrosinated tubulin (or Glu tubulin after its C-terminus), which accumulates in MTs after they are stabilized (Webster et al., 1987; Khawaja et al., 1988). We then scored cells for their content of Glu MTs. Cells containing five or more brightly stained, extended Glu MTs were counted as positive for Glu MTs. For MTOC reorientation, we assessed the position of the MTOC (detected with antibodies to -tubulin) relative to the leading edge and nucleus, scoring MTOCs as reoriented if they were in a pie-shaped segment facing the wound; random orientation of the MTOC gives a value 33% "reoriented" by this method (see Palazzo et al., 2001b).

Because LPA stimulates inhibition of GSK3 (Fang et al., 2002; and see below), we first tested whether known inhibitors of GSK3, LiCl, or SB216763 mimicked the effect of LPA treatment of starved fibroblasts. When starved fibroblasts were treated with either GSK3 inhibitor, the level of Glu MTs increased without significantly affecting the levels of the bulk, dynamic MTs detected with Tyr tubulin antibody (Figure 1, A and B). As with LPA treatment, Glu MTs induced by the GSK3 inhibitors constituted a subset of the total MTs. However, unlike those triggered by LPA, they were not preferentially oriented toward the wound edge (Figure 1A). Glu MTs induced by GSK3 inhibitors were not noticeably bundled, indicating that MAP-like proteins were not involved. SB216763 treatment of starved NIH 3T3 fibroblasts also promoted an increase in nocodazole-resistant MTs (Figure 1C), indicating that inhibition of GSK3 affects MT stability and not the enzymes responsible for tubulin post-translational modifications. These results show that inhibition of GSK3 is sufficient to trigger the formation of stabilized MTs in fibroblasts.

View larger version (67K): [in this window] [in a new window]   Figure 1. GSK3 negatively regulates LPA-induced stable MT formation. (A) Distribution of Glu and Tyr MTs in wounded, starved NIH 3T3 fibroblasts left untreated or treated with 5 µM LPA, 20 mM LiCl, 2 µM SB216763, or microinjected with DNA encoding GSK3 (arrow) and then treated with LPA (inset shows GSK3 expression in the injected cell). After 2 h, cells were fixed and immunostained for Glu and Tyr tubulin to monitor changes in stable and bulk MTs, respectively. Note that Glu MTs induced by LiCl or SB216763 were not oriented toward the wound-edge as were those induced by LPA. (B) Quantification of percentage of wounded, starved NIH 3T3 fibroblasts with Glu MTs after the indicated treatments (untr, untreated). Data are mean ± SEM from at least three experiments (n >70 cells). (C) Nocodazole-resistant microtubules in wounded, starved NIH 3T3 fibroblasts treated with or without 2 µM SB216763 for 2 h. Nocodazole resistance was assessed by treating cells with 2 µM nocodazole for 30 min, detergent extracting, and fixing before immunostaining for -tubulin. Scale bars, (A and C) 20 µm.

GSK3 Does Not Affect LPA-stimulated MTOC Reorientation
To test whether GSK3 was involved in MTOC reorientation in fibroblasts, we treated wounded, starved NIH 3T3 fibroblast monolayers with SB216763 in the absence or presence of LPA to promote MTOC reorientation. SB216763 did not induce MTOC reorientation in starved cells and did not inhibit MTOC reorientation induced by LPA (Figure 2, A and B). To extend this finding, starved NIH 3T3 fibroblasts were microinjected with wild-type GSK3 in the presence or absence of LPA to examine whether overexpression of GSK3 inhibited MTOC reorientation. Expression of wild-type GSK3, which inhibited MT stabilization induced by LPA (Figures 1, A and B, and 2A), did not inhibit MTOC reorientation induced by LPA or promote MTOC reorientation on its own (Figure 2, A and B). Thus, in starved NIH 3T3 fibroblasts, GSK3 is involved in regulating MT stabilization but not MTOC reorientation. This is consistent with our previous studies showing that factors working in one of the MT regulatory pathways in fibroblasts do not function in the other pathway (Palazzo et al., 2001b; Wen et al., 2004).

View larger version (57K): [in this window] [in a new window]   Figure 2. GSK3 does not affect MTOC reorientation. (A) Merged images depicting Glu tubulin (red), -tubulin (green), and DAPI staining (blue) of wounded, starved NIH 3T3 fibroblasts treated 10 µM SB216763 for 1–2 h or microinjected with plasmid encoding GSK3 (arrows) and either left untreated (–LPA) or treated with 5 µM LPA (+LPA) for 2 h (insets show the cells expressing GSK3). Wound edge is toward the top in each panel and is depicted with a dashed white line. (B) Quantification of percentage of wounded, starved NIH 3T3 fibroblasts with oriented MTOCs after the indicated treatments. Data are mean ± SEM from at least three experiments (n >40 cells). Random distribution of the MTOC would lead to 33% of the cells with oriented MTOCs. Scale bar, (A) 20 µm.

GSK3 Functions in the Rho-mDia-EB1-APC MT Stabilization Pathway

View larger version (78K): [in this window] [in a new window]   Figure 3. GSK3 functions between Rho and EB1 in the MT stabilization pathway. (A) Microinjected C3 toxin does not inhibit LiCl-induced Glu MTs, whereas expression of microinjected EB1C-GFP DNA does inhibit LiCl-induced Glu MTs. C3 was coinjected with human IgG as an injection marker. Cells were fixed and stained for actin, and Tyr and Glu MTs. Arrows indicate injected cells. (B) Quantification of percentage of wounded, starved NIH 3T3 fibroblasts with Glu MTs after the indicated treatments (untr, untreated). Data are mean ± SEM from at least three experiments (n >50 cells). (C) LiCl does not induce changes in actin organization. Starved, wounded NIH 3T3 cells were left untreated or treated with either 20 mM LiCl or 5 µM LPA for 90 min, fixed, and stained for F-actin with rhodamine-phalloidin. Scale bars, (A and C) 20 µm.

mDia Is Required for the LPA-stimulated Phosphorylation of GSK3
LPA treatment of starved Swiss 3T3 fibroblasts was shown earlier to increase GSK3 Ser9 phosphorylation, although the mechanism for this was not fully delineated (Fang et al., 2002). We confirmed that LPA treatment of starved COS-7 cells or NIH 3T3 fibroblasts increased GSK3 pSer9 levels (Figure 4, A and B). In both cases, the levels of GSK3 pSer9 triggered by LPA was similar to that induced by insulin, which stimulates GSK3 pSer9 through a pathway involving PKB/Akt (Cross et al., 1995).

View larger version (74K): [in this window] [in a new window]   Figure 4. mDia is required for GSK3 phosphorylation and MT stabilization induced by LPA. (A) Western blot of starved COS-7 cells transfected with HA-tagged GSK3 and either left untreated, treated with 10 µg/ml insulin, 10 µM LPA (both for 5 min) or cotransfected with GFP-tagged GBD-mDia constructs. Blots were probed with antibody to GSK3 pSer9 (top) or total GSK3 (bottom). Positions of HA-GSK3 pSer9, expressed HA-GSK3 and endogenous GSK3/ are shown on the left. (B) Western blot of endogenous GSK3 pSer9 levels in starved NIH 3T3 cells either left untreated, treated with 10 µg/ml insulin, 10 µM LPA (both for 5 min), or transfected with GFP alone, GFP-521-1040-mDia2, or GFP-DAD. Blots were probed with antibody to GSK3 pSer9 (top) or total GSK3 (bottom). Positions of endogenous GSK3/ are shown on the left. (C) Western blots of COS-7 cells treated as in A, except that siRNA specific for mDia1 or GFP was cotransfected along with HA-GSK3. Western blots were probed with antibodies to mDia1, GSK3 pSer9, total GSK3 and vinculin (as a loading control). Mock-treated cells received transfection reagents alone. (D) Quantification of average fluorescent intensity of GSK3 pSer9 in immunofluorescently stained cells that had been mock-transfected, or transfected with mDia1 or GAPDH siRNA. Cells were left untreated (untr) or stimulated with 10 µg/ml insulin or 10 µM LPA (both for 5 min) as indicated. Average fluorescent intensity is expressed as the percentage of mock-transfected, serum-starved cells. *p < 0.01 compared with mock LPA; **p < 0.05 compared with mock insulin by Student's t test. Data are mean ± SEM from three experiments in which >70 cells were measured. (E) Immunofluorescence images of starved NIH 3T3 fibroblasts microinjected with 10 µM mDia1 or GAPDH siRNAs and FITC-dextran as an injection marker. Cells were serum-starved for 40–48 h after injection and then treated with 5 µM LPA (90 min) before fixation and staining for mDia1 (or GAPDH), Glu, and Tyr MTs. Arrows indicate siRNA-injected cells with reduced levels of mDia1 or GAPDH. (F) Quantification of percentage of wounded, starved NIH 3T3 fibroblasts with Glu MTs after transfection with labeled mDia1 or GAPDH siRNA. Cells were stimulated with LPA (5 µM, 90 min) as indicated. *p < 0.001 compared with mock LPA. Data are mean ± SEM from three experiments. n >180 cells. Scale bar, (E) 20 µm.

Next, we determined whether activation of endogenous mDia1 was sufficient to stimulate endogenous GSK3 phosphorylation on Ser9. NIH 3T3 cells were transfected with GFP-tagged Dia autoregulatory domain (GFP-DAD), a fragment of mDia1 that is able to activate the endogenous protein by relieving autoinhibition (Alberts, 2001). Transfection with DAD was sufficient to promote phosphorylation of endogenous GSK3 in NIH 3T3 cells, whereas the control transfection with GFP alone had no effect (Figure 4B). In addition, expression of GFP-521-1040-mDia2, which contains the FH1 and FH2 domains and is the minimal domain of mDia that is sufficient to stimulate stable MT formation (Wen et al., 2004), was also sufficient to promote GSK3 pSer9 in NIH3T3 cells (Figure 4B).

To test whether endogenous mDia1 was required for the LPA-stimulated increase in GSK3 pSer9 levels, we knocked down mDia1 with siRNA. COS-7 cells were transfected with HA-GSK3 as before, in combination with siRNA specific for mDia1 or control siRNA specific for GFP. mDia1 siRNA decreased endogenous mDia1 by 75%, whereas the control siRNA against GFP did not affect mDia1 levels (Figure 4C). mDia1 siRNA, but not GFP siRNA, reduced LPA-stimulated HA-GSK3 pSer9 by 50%, but did not affect basal HA-GSK3 pSer9 levels in unstimulated cells or the induction of HA-GSK3 pSer9 in cells stimulated with insulin (Figure 4C). These data demonstrate that mDia1 is responsible for at least part of the enhanced GSK3 pSer9 levels induced by LPA and that mDia1 does not participate in insulin-stimulated regulation of GSK3, which occurs through the PKB/Akt pathway (Cross et al., 1995).

To quantify the effect of mDia1 knockdown on endogenous Ser9 phosphorylation of GSK3 in NIH 3T3 cells, a quantitative immunofluorescence approach was taken because the transfection efficiency was not sufficient to permit a Western blot assay. NIH 3T3 were transfected with siRNA to mDia1, or siRNA to GAPDH as a control, serum-starved, and then treated with LPA or insulin. Cells were fixed and stained for GSK3 pSer9, and mDia1 or GAPDH. The average fluorescence intensity per cell was measured in cells stained for GSK3 pSer9 that had visible mDia1 (or GAPDH) knockdown (for examples, see Figure 4E). This assay showed a consistent 30–50% increase in GSK3 pSer9 fluorescence in LPA- and insulin-treated cells, demonstrating the validity of this assay (Figure 4D). Knockdown of mDia1, but not GAPDH, resulted in a significant decrease in LPA-stimulated GSK3 pSer9 (Figure 4D). There was also a decrease in insulin-stimulated GSK3 pSer9 in mDia1 siRNA-treated cells, but this decrease was less significant compared with LPA (Figure 4D). These data demonstrate that mDia1 is required for at least some of the enhanced GSK3-pSer9 levels induced by LPA.

mDia1 Is Required for Stable MT Formation
Previous studies identified mDia as the likely Rho effector for LPA-mediated MT stabilization (Palazzo et al., 2001a). Given that mDia1 is required for LPA-induced increase in GSK3 pSer9 and that GSK3 appeared to be involved in the MT stabilization pathway, it was important to confirm that mDia was necessary for MT stabilization induced by LPA as previously reported (Goulimari et al., 2005). Wound-edge starved NIH 3T3 fibroblasts, which express mDia1 but not mDia2 (Tominaga et al., 2000; and unpublished data), were microinjected with siRNA against mDia1 that we had validated in COS-7 cells (Figure 4C). Two days after mDia1 siRNA treatment or GAPDH siRNA as a control, the cells were treated with LPA to stimulate formation of stable MTs. The majority of cells injected with siRNA had reduced levels of mDia1 or GAPDH as detected by immunofluorescence, confirming the efficacy of the knockdown in NIH 3T3 fibroblasts (Figure 4E). In cells with visible knockdown of mDia1, there was a decrease in the level of Glu MTs in response to LPA compared with uninjected cells or GAPDH siRNA injected cells (Figure 4E). Quantification of this effect in cells transfected with labeled siRNA (to identify the transfected cells) showed that mDia1 knockdown significantly reduced the induction of Glu MTs by LPA compared with untransfected or siRNA GAPDH controls (Figure 4F).

Novel PKCs Are Required for GSK3 Phosphorylation and MT Stabilization by LPA
LPA-stimulated GSK3 pSer9 is blocked by PKC inhibitors (Fang et al., 2002; Yang et al., 2005), and we confirmed this result in NIH 3T3 and COS-7 cells, where treatment with the general PKC inhibitor, bisindolylmaleimide I, strongly inhibited LPA-stimulated, but not insulin-stimulated, GSK3 pSer9 (Figure 5, A and B). Next, we tested whether mDia-stimulated GSK3 pSer9 also required PKC. COS-7 cells transfected with HA-GSK3 were pretreated with bisindolylmaleimide I and then treated with LPA or insulin or cotransfected with constitutively active GBD-mDia2, and their levels of GSK3 pSer9 were assayed. GSK3 pSer9 induced by LPA or active mDia2 was inhibited by bisindolylmaleimide I, indicating that LPA/mDia-induced GSK3 pSer9 required PKC(s) (Figure 5B). Insulin-stimulated GSK3 pSer9 was less affected, as expected (Figure 5B).

View larger version (49K): [in this window] [in a new window]   Figure 5. Novel PKCs are involved in GSK3 phosphorylation and MT stabilization. (A) Western blot of starved NIH 3T3 cells left untreated (untr) or treated with 100 µg/ml insulin or 10 µM LPA for 5 min. Some cells were pretreated with 10 µM bisindolylmaleimide I (BIM-1) for 1 h before stimulation. (B) Western blot of COS-7 cells transfected with HA-tagged GSK3 and either cotransfected with GFP-tagged GBD-mDia2 or treated with 100 µg/ml insulin or 10 µM LPA for 5 min after 24-h serum starvation. Some cells were treated with 10 µM bisindolylmaleimide I (BIM-1) for 1 h before stimulation. Blots were probed with antibodies to GSK3 pSer9 or total GSK3. The position of HA-GSK3 pSer9, HA-GSK3, and endogenous GSK3/ are indicated on the left. (C) Immunofluorescence images of Glu and Tyr MTs in wounded, starved NIH 3T3 fibroblasts expressing microinjected dominant negative HA-tagged PKC constructs (K to R mutations in the ATP binding domain) and treated with 2 µM LPA for 90 min. Arrows show expressing cells; insets show HA staining. (D) Quantification of percentage of wounded, starved NIH 3T3 fibroblasts with Glu MTs after expression of microinjected DNA encoding dominant negative HA-tagged PKC isoforms and treatment with 2 µM LPA for 90 min. Results are mean ± SEM from at least three experiments. n 30 cells. (E) Quantification of percentage of wounded, starved NIH 3T3 fibroblasts with oriented MTOCs after the indicated treatments. Data are mean ± SEM from at least three experiments. n 20 cells. Random distribution of the MTOC would lead to 33% of the cells with oriented MTOCs. (F) Western blots of COS-7 cells transfected with GFP-tagged GSK3 and either cotransfected with pcDNA3 (as a control) or HA-tagged dominant negative PKC (HA-PKC-KR), and myc-GFP-tagged GBD-mDia1. After 24-h transfection and 24-h serum-starvation, cells were left untreated or treated with 10 µg/ml insulin or 10 µM LPA for 10 min. Blots were probed with antibodies to GSK3 pSer9, total GSK3, and HA tags to show expression of the dominant negative HA-PKC. (G) Quantification of average fluorescence intensity of GSK3 pSer9 in immunofluorescently stained cells that had been transfected with GFP-GBD-mDia1, HA-PKC-KR, or both. Cells were stimulated with 10 µg/ml insulin or 10 µM LPA (both for 5 min) as indicated. Average fluorescence intensity is expressed as the percentage of untransfected, serum-starved cells. *p < 0.05 compared with untreated; **p < 0.05 compared with GBD-mDia1; ***p < 0.01 compared with LPA; +p > 0.05 compared with insulin by Student's t test. Data are mean ± SEM from at least three experiments. n >70 cells. Bar, (C) 20 µm.

To test whether novel PKCs were sufficient to promote stable MTs, plasmids encoding the isolated catalytic (cat) domain were expressed in starved cells. Expression of PKC-cat or PKC-cat did not induce stable MTs in the absence of LPA treatment (unpublished data). Thus, although novel PKCs are required for the formation of stable MTs induced by LPA, their catalytic activity alone is not sufficient.

To check whether the novel PKCs affected GSK3 pSer9, we analyzed the ability of the dominant negative constructs to inhibit LPA- and mDia-induced GSK3 pSer9. Coexpression of dominant negative PKC in COS-7 cells with GFP-GSK3 as a reporter construct inhibited the increase in GSK3 pSer9 levels in response to LPA and GBD-mDia1, but not insulin (Figure 5F). Thus, dominant negative PKC that inhibits MT stabilization also inhibits GSK3 phosphorylation.

To quantify the effect of dominant negative PKC on endogenous GSK3 pSer9, we again performed quantitative immunofluorescence to measure GSK3 pSer9. COS-7 cells were transfected with dominant negative PKC, GFP-GBD-mDia1, or both, serum-starved, and then treated with LPA or insulin as indicated. Cells were fixed and stained, and individual cells expressing one or both proteins were measured for their levels of GSK3 pSer9. Again, this assay showed a consistent increase in GSK3 pSer9 fluorescence in LPA- and insulin-treated cells, similar to what was observed in NIH 3T3 cells (Figure 5G). In addition, transfection with active mDia1 significantly increased endogenous GSK3 pSer9 over levels in untreated cells, as had been observed for transfected GSK3. Expression of PKC-KR significantly reduced the GBD-mDia1 and LPA stimulation of GSK3 pSer9 to background levels (Figure 5G). PKC-KR also decreased GSK3 pSer9 stimulation due to insulin, but this decrease was not significant and did not reduce the levels to those of untreated cells (Figure 5G).

mDia Interacts Directly with Novel PKCs
The above results raise the possibility that mDia acts in conjunction with novel PKC isoforms to phosphorylate and inhibit GSK3. To explore whether this might be a direct effect of mDia on novel PKCs, we tested whether the two proteins interacted in vivo. Mouse brain extract was immunoprecipitated with antibody to PKC. A small amount of mDia1 was visualized in the immunoprecipitate, demonstrating that PKC and mDia1 interact at physiological levels (Figure 6A). Because we found that only novel PKC isoforms specifically affected stable microtubules, we examined whether other isoforms of PKC were capable of binding to mDia. Tagged versions of active mDia1 and full-length PKC isoforms were transfected into COS-7 cells and immunoprecipitated. Immunoprecipitation of myc-GFP-GBD-mDia1, but not myc-GFP alone, coimmunoprecipitated the novel PKC isoforms, PKC and PKC, and also the atypical PKC isoform, PKC (Figure 6B). Another HA-tagged kinase, ROK, did not coimmunoprecipitate with mDia1. Thus, active mDia1 is capable of binding multiple PKC isoforms in vivo when overexpressed.

View larger version (27K): [in this window] [in a new window]   Figure 6. mDia interacts with novel PKC isoforms in vivo and in vitro. (A) Mouse brain extract was immunoprecipitated with antibody to PKC or an equal amount of rabbit IgG as a control. Bound proteins were eluted and analyzed by Western blotting with antibodies against PKC and mDia1. The input (2%) of each reaction is depicted in the left panels. (B) Western blots of COS-7 cells cotransfected with myc-GFP-GBD-mDia1 (or myc-GFP as a control) and HA-tagged full-length PKCs or ROK. After 24-h expression, cell lysates were prepared and immunoprecipitated with GFP antibody. Western blots of immunoprecipitates were probed with antibody against HA or myc. (C) Equimolar amounts of PKC were electrophoresed and transferred to nitrocellulose. The membrane was Ponceau-stained to confirm equal loading. The membrane then was incubated with 10 µg/ml GST-521-1040-mDia2, and the mDia2 bound to PKCs was detected with an antibody to GST. The amount of bound mDia2 was quantified, and the ratio of PKC to PKC binding was calculated from three independent experiments. (D) Purified recombinant PKC or PKC was added to equimolar amounts of GST or GST-521-1040-mDia2 prebound to glutathione-agarose beads in buffer with and without phosphatidylserine and diacylglycerol (±lipids). Bound proteins were eluted and analyzed by Western blotting with antibody against PKC or PKC. Five percent of the input was analyzed by Western blotting with antibody to PKC, PKC, or GST to determine equal loading.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We demonstrate that GSK3 functions and is phosphorylated in the Rho-mDia mediated MT stabilization pathway, acting downstream of Rho and upstream of EB1. GSK3 is negatively regulated by signals that are sufficient to promote MT stabilization, because knockdown of both mDia1 and dominant negative novel PKCs block MT stabilization and GSK3 phosphorylation. These data provide a novel role for a formin protein in regulating the action of a kinase, although earlier work showed mDia could interact with Src kinase (Tominaga et al., 2000). Recent studies of formins in mammalian cells have focused on their effects on the actin and MT cytoskeletons (reviewed in Wallar and Alberts, 2003; Zigmond, 2004). Here, we demonstrate that LPA, through mDia1, can regulate the activity of GSK3 by controlling its phosphorylation.

Our results point to novel PKCs as the likely candidates for mediating the effects of mDia on GSK3. We found that dominant negative novel PKCs blocked active mDia induced GSK3 pSer9 and that mDia interacted directly with PKC. Although mDia also bound to another PKC isoform, PKC, the affinity of PKC was higher for mDia and could account for the specific activity of novel PKCs toward stable MTs. Furthermore, although the binding of PKC to mDia was enhanced in the presence of lipids, the binding of PKC was not. These results suggest that binding of active mDia to novel PKCs may regulate their activity toward substrates such as GSK3. Previous work showed that novel PKCs can phosphorylate GSK3 on Ser9 (Fang et al., 2002). We suggest that mDia may regulate the activity or localization of novel PKCs to enhance their activity toward GSK3. This LPA-stimulated pathway involving mDia and novel PKC regulation of GSK3 functions independently of insulin-PKB regulation of GSK3.

This work identifies two new components, novel PKCs and GSK3 that function in the Rho-mDia-EB1-APC pathway, which regulates plus-end mediated stabilization of MTs. Our work points to an new role for novel PKCs in governing MT stabilization. Novel PKCs are characterized by their dependence on lipids for activation, but they do not require calcium. Novel PKCs (, , , and ) are widely and differentially expressed in tissues and each isoform has unique functions. We do not know how mDia binding to PKC may regulate its activity. Perhaps the binding of PKC to mDia brings it into the vicinity of MTs, where it is sometimes localized in vivo (Kato et al., 2001; Palazzo et al., 2001a; Wen et al., 2004). PKC is also localized to the actin cytoskeleton in some cells (Prekeris et al., 1996; Hernandez et al., 2001), and it is also possible that mDia regulates its activity toward the actin cytoskeleton.

GSK3 is the first negative regulator to be identified in the Rho-mDia-EB1-APC pathway for MT stabilization. A number of earlier studies have used inhibitors to show that GSK3 regulates MT stability (Goold et al., 1999; Krylova et al., 2000; Akhmanova et al., 2001). Our work extends these studies by showing how a known MT regulatory pathway controls GSK3 phosphorylation in response to a physiological stimulus. Most previous studies have implicated GSK3 in regulating MT stability through its affects on MAPs or CLASPs binding laterally to MTs (Lovestone et al., 1996; Wagner et al., 1996; Goold et al., 1999; Krylova et al., 2000; Akhmanova et al., 2001). Our results indicate that GSK3 also regulates MT stability through its action on factors involved in MT capture at the cell cortex. Glu MTs are a stabilized subset of MTs that are capped at their distal ends, preventing subunit addition or loss and their ends are found in close proximity to the cell cortex (Infante et al., 2000; Wen et al., 2004). Direct interaction of mDia with the MT tip proteins EB1 and APC contributes to this end-mediated MT stabilization (Wen et al., 2004). The interaction of these proteins with each other or with MTs are potential targets for regulation by GSK3. The only known substrate of GSK3 among these proteins is APC, and GSK3 phosphorylation of APC decreases its interaction with MTs in vitro (Zumbrunn et al., 2001). APC interaction with EB1 is also regulated by phosphorylation, although this is by other kinases (Askham et al., 2000). It will be interesting to test whether other interactions among these proteins and with MTs are regulated by GSK3 phosphorylation. GSK3 also appears to negatively regulate the interaction of CLASPs with microtubules (Akhmanova et al., 2001; Wittmann and Waterman-Storer, 2005), so this is another candidate target for GSK3.

The observation that inhibition of GSK3 induces stable MTs that, in contrast to LPA-stimulated stable MTs, are not polarized toward the leading edge, is likely due to the global inactivation caused by GSK3 inhibitors and the fact that GSK3 functions downstream of Rho. Selective stabilization of MTs oriented toward the wound edge occurs because of integrin engagement in the front of the cell, leading to activation of FAK (Palazzo et al., 2004). This adhesion signal is thought to restrict the ability of active Rho to stimulate mDia to the region near the leading edge. Consistent with this idea, overexpression of active Rho promotes the formation of a polarized array of stable MTs, but overexpression of constitutively active mDia does not (Cook et al., 1998; Palazzo et al., 2001a). GSK3 bypasses the adhesion signal that polarizes the newly formed stable MTs by acting downstream of mDia. Indeed, we have found that GSK3 inhibition by LiCl is able to promote MT stabilization in nonadherent fibroblasts that lack integrin signals (Palazzo and Gundersen, unpublished observations).

The MT array is also polarized in migrating cells by reorientation of the MTOC to a position between the nucleus and the leading edge of the cell (Gotlieb et al., 1981; Kupfer et al., 1982; Gundersen and Bulinski, 1988). We previously found that inhibition of Cdc42, dynein, or dynactin, proteins involved in MTOC reorientation, does not block MT stabilization and that inhibition of Rho or EB1, proteins involved in MT stabilization, does not block MTOC reorientation (Palazzo et al., 2001b; Wen et al., 2004). We find that GSK3 and novel PKCs function in the MT stabilization pathway, which is consistent with these results, but not in the MTOC reorientation pathway in NIH 3T3 fibroblasts and that atypical PKCs function in MTOC reorientation, as reported previously (Etienne-Manneville and Hall, 2003; Gomes et al., 2005), but not in MT stabilization. That inhibition of GSK3 is both necessary and sufficient for MT stabilization, but not for MTOC reorientation, is in contrast to results in wounded astrocytes, where GSK3 overexpression or inhibition has been found to inhibit MTOC reorientation (Etienne-Manneville and Hall, 2003). A role for GSK3 in MT stabilization in astrocytes has not yet been tested. We do not know the reason for the different effects of GSK3 in the two systems, but they may be related to differences in cell types, conditions used to stimulate MTOC reorientation, or to the much longer time to reorient MTOCs in astrocytes (6 h) compared with fibroblasts (1 h).

Despite the differences between the responses of different cell types to GSK3, it is clear that GSK3 is an important regulator of microtubule polarity, and thus a key determinant of polarity during development and differentiation. GSK3 has a crucial role in Wnt signaling involving APC, Dvl, Axin, and -catenin, which mediates embryonic polarity. Recent studies in neurons have also pointed to the significance of GSK3 in the establishment of axons and maintenance of neuronal polarity via APC, PI3K, and CRMP-2 (Shi et al., 2004; Jiang et al., 2005; Yoshimura et al., 2005). Whether the establishment of polarity by GSK3 in these situations requires the formation of stable microtubules will be an interesting question to pursue.

	ACKNOWLEDGMENTS

 
We thank J. W. Soh, A. Alberts, D. Brautigan, J. Kitwajewski, P. Scheiffle, C. Carpenter, and F. Bartolini for DNA constructs; Y. Wen for preparation of GST protein; and members of the Gundersen lab for comments on the manuscript. C.H.E. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship. This work was supported by National Institutes of Health Grant GM 62939 (G.G.G.).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-10-0914) on September 20, 2006.

Address correspondence to: Gregg G. Gundersen (ggg1{at}columbia.edu)

Abbreviations used: GSK3, glycogen synthase kinase 3; PKC, protein kinase C; MTs, microtubules; MTOC, microtubule organizing center; LPA, lysophosphatidic acid; pSer9, phosphorylated serine 9

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Akhmanova, A., et al. (2001). CLASPS are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts. Cell 104, 923–935.[CrossRef][Medline]

Aktories, K., Braun, U., Rosener, S., Just, I., Hall, A. (1989). The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. Biochem. Biophys. Res. Commun 158, 209–213.[CrossRef][Medline]

Alberts, A. S. (2001). Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain. J. Biol. Chem 276, 2824–2830.[Abstract/Free Full Text]

Arakawa, Y., Bito, H., Furuyashiki, T., Tsuji, T., Takemoto-Kimura, S., Kimura, K., Nozaki, K., Hashimoto, N., Narumiya, S. (2003). Control of axon elongation via an SDF-1alpha/Rho/mDia pathway in cultured cerebellar granule neurons. J. Cell Biol 161, 381–391.[Abstract/Free Full Text]

Askham, J. M., Moncur, P., Markham, A. F., Morrison, E. E. (2000). Regulation and function of the interaction between the APC tumour suppressor protein and EB1. Oncogene 19, 1950–1958.[CrossRef][Medline]

Bulinski, J. C. and Gundersen, G. G. (1991). Stabilization of post-translational modification of microtubules during cellular morphogenesis. Bioessays 13, 285–293.[CrossRef][Medline]

Coghlan, M. P., Chou, M. M., Carpenter, C. L. (2000). Atypical protein kinases Clambda and -zeta associate with the GTP-binding protein Cdc42 and mediate stress fiber loss. Mol. Cell. Biol 20, 2880–2889.[Abstract/Free Full Text]

Cook, T. A., Nagasaki, T., Gundersen, G. G. (1998). Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid. J. Cell Biol 141, 175–185.[Abstract/Free Full Text]

Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789.[CrossRef][Medline]

Erck, C., et al. (2005). A vital role of tubulin-tyrosine-ligase for neuronal organization. Proc Natl. Acad. Sci. USA 102, 7853–7858.[Abstract/Free Full Text]

Etienne-Manneville, S. and Hall, A. (2001). Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKC. Cell 106, 489–498.[CrossRef][Medline]

Etienne-Manneville, S. and Hall, A. (2003). Cdc42 regulates GSK-3 and adenomatous polyposis coli to control cell polarity. Nature 421, 753–756.[CrossRef][Medline]

Fang, X., Yu, S., Tanyi, J. L., Lu, Y., Woodgett, J. R., Mills, G. B. (2002). Convergence of multiple signaling cascades at glycogen synthase kinase 3, Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway. Mol. Cell. Biol 22, 2099–2110.[Abstract/Free Full Text]

Gomes, E. R., Jani, S., Gundersen, G. G. (2005). Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 121, 451–463.[CrossRef][Medline]

Goold, R. G., Owen, R., Gordon-Weeks, P. R. (1999). Glycogen synthase kinase 3beta phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones. J. Cell Sci 112, Pt 193373–3384.[Abstract]

Gotlieb, A. I., May, L. M., Subrahmanyan, L., Kalnins, V. I. (1981). Distribution of microtubule organizing centers in migrating sheets of endothelial cells. J. Cell Biol 91, 589–594.[Abstract/Free Full Text]

Goulimari, P., Kitzing, T. M., Knieling, H., Brandt, D. T., Offermanns, S., Grosse, R. (2005). Galpha12/13 is essential for directed cell migration and localized Rho-Dia1 function. J. Biol. Chem 280, 42242–42251.[Abstract/Free Full Text]

Gundersen, G. G. and Bulinski, J. C. (1986). Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin. Eur. J. Cell Biol 42, 288–294.[Medline]

Gundersen, G. G. and Bulinski, J. C. (1988). Selective stabilization of microtubules oriented toward the direction of cell migration. Proc. Natl. Acad. Sci. USA 85, 5946–5950.[Abstract/Free Full Text]

Gundersen, G. G., Gomes, E. R., Wen, Y. (2004). Cortical control of microtubule stability and polarization. Curr. Opin. Cell Biol 16, 106–112.[CrossRef][Medline]

Gundersen, G. G., Kalnoski, M. H., Bulinski, J. C. (1984). Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell 38, 779–789.[CrossRef][Medline]

Gundersen, G. G., Kim, I., Chapin, C. J. (1994). Induction of stable microtubules in 3T3 fibroblasts by TGF-beta and serum. J. Cell Sci 107, Pt 3645–659.[Abstract]

Gurland, G. and Gundersen, G. G. (1995). Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts. J. Cell Biol 131, 1275–1290.[Abstract/Free Full Text]

Hernandez, R. M., Wescott, G. G., Mayhew, M. W., McJilton, M. A., Terrian, D. M. (2001). Biochemical and morphogenic effects of the interaction between protein kinase C-epsilon and actin in vitro and in cultured NIH3T3 cells. J. Cell. Biochem 83, 532–546.[CrossRef][Medline]

Infante, A. S., Stein, M. S., Zhai, Y., Borisy, G. G., Gundersen, G. G. (2000). Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap. J. Cell Sci 113, 3907–3919.[Abstract]

Jiang, H., Guo, W., Liang, X., Rao, Y. (2005). Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators. Cell 120, 123–135.[Medline]

Kato, T., Watanabe, N., Morishima, Y., Fujita, A., Ishizaki, T., Narumiya, S. (2001). Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells. J. Cell Sci 114, 775–784.[Abstract]

Khawaja, S., Gundersen, G. G., Bulinski, J. C. (1988). Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level. J. Cell Biol 106, 141–149.[Abstract/Free Full Text]

Kodama, A., Karakesisoglou, I., Wong, E., Vaezi, A., Fuchs, E. (2003). ACF7, an essential integrator of microtubule dynamics. Cell 115, 343–354.[CrossRef][Medline]

Kreitzer, G., Liao, G., Gundersen, G. G. (1999). Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism. Mol. Biol. Cell 10, 1105–1118.[Abstract/Free Full Text]

Krylova, O., Messenger, M. J., Salinas, P. C. (2000). Dishevelled-1 regulates microtubule stability: a new function mediated by glycogen synthase kinase-3beta. J. Cell Biol 151, 83–94.[Abstract/Free Full Text]

Kupfer, A., Dennert, G., Singer, S. J. (1983). Polarization of the Golgi apparatus and the microtubule-organizing center within cloned natural killer cells bound to their targets. Proc. Natl. Acad. Sci. USA 80, 7224–7228.[Abstract/Free Full Text]

Kupfer, A., Louvard, D., Singer, S. J. (1982). Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound. Proc. Natl. Acad. Sci. USA 79, 2603–2607.[Abstract/Free Full Text]

Leung, T., Chen, X. Q., Manser, E., Lim, L. (1996). The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol 16, 5313–5327.[Abstract]

Liao, G. and Gundersen, G. G. (1998). Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. J. Biol. Chem 273, 9797–9803.[Abstract/Free Full Text]

Lin, S. X., Gundersen, G. G., Maxfield, F. R. (2002). Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (glu) microtubules and kinesin. Mol. Biol. Cell 13, 96–109.[Abstract/Free Full Text]

Lovestone, S., Hartley, C. L., Pearce, J., Anderton, B. H. (1996). Phosphorylation of tau by glycogen synthase kinase-3 beta in intact mammalian cells: the effects on the organization and stability of microtubules. Neuroscience 73, 1145–1157.[CrossRef][Medline]

Naranatt, P. P., Krishnan, H. H., Smith, M. S., Chandran, B. (2005). Kaposi's sarcoma-associated herpes virus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus. J. Virol 79, 1191–1206.[Abstract/Free Full Text]

Palazzo, A. F., Cook, T. A., Alberts, A. S., Gundersen, G. G. (2001a). mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat. Cell Biol 3, 723–729.[CrossRef][Medline]

Palazzo, A. F., Eng, C. H., Schlaepfer, D. D., Marcantonio, E. E., Gundersen, G. G. (2004). Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling. Science 303, 836–839.[Abstract/Free Full Text]

Palazzo, A. F., Joseph, H. L., Chen, Y. J., Dujardin, D. L., Alberts, A. S., Pfister, K. K., Vallee, R. B., Gundersen, G. G. (2001b). Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr. Biol 11, 1536–1541.[CrossRef][Medline]

Prekeris, R., Mayhew, M. W., Cooper, J. B., Terrian, D. M. (1996). Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function. J. Cell Biol 132, 77–90.[Abstract/Free Full Text]

Ridley, A. J. and Hall, A. (1992). The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389–399.[CrossRef][Medline]

Shi, S. H., Cheng, T., Jan, L. Y., Jan, Y. N. (2004). APC and GSK-3beta are involved in mPar3 targeting to the nascent axon and establishment of neuronal polarity. Curr. Biol 14, 2025–2032.[CrossRef][Medline]

Soh, J. W., Lee, E. H., Prywes, R., Weinstein, I. B. (1999). Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol. Cell. Biol 19, 1313–1324.[Abstract/Free Full Text]

Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Courtneidge, S. A., Alberts, A. S. (2000). Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling. Mol. Cell 5, 13–25.[CrossRef][Medline]

Tzima, E., Kiosses, W. B., del Pozo, M. A., Schwartz, M. A. (2003). Localized cdc42 activation, detected using a novel assay, mediates microtubule organizing center positioning in endothelial cells in response to fluid shear stress. J. Biol. Chem 278, 31020–31023.[Abstract/Free Full Text]

Wagner, U., Utton, M., Gallo, J. M., Miller, C. C. (1996). Cellular phosphorylation of tau by GSK-3 beta influences tau binding to microtubules and microtubule organisation. J. Cell Sci 109, Pt 61537–1543.[Abstract]

Wallar, B. J. and Alberts, A. S. (2003). The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol 13, 435–446.[CrossRef][Medline]

Watanabe, N., Madaule, P., Reid, T., Ishizaki, T., Watanabe, G., Kakizuka, A., Saito, Y., Nakao, K., Jockusch, B. M., Narumiya, S. (1997). p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J 16, 3044–3056.[CrossRef][Medline]

Webster, D. R., Gundersen, G. G., Bulinski, J. C., Borisy, G. G. (1987). Differential turnover of tyrosinated and detyrosinated microtubules. Proc. Natl. Acad. Sci. USA 84, 9040–9044.[Abstract/Free Full Text]

Wen, Y., Eng, C. H., Schmoranzer, J., Cabrera-Poch, N., Morris, E. J., Chen, M., Wallar, B. J., Alberts, A. S., Gundersen, G. G. (2004). EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat. Cell Biol 6, 820–830.[CrossRef][Medline]

Wittmann, T. and Waterman-Storer, C. M. (2005). Spatial regulation of CLASP affinity for microtubules by Rac1 and GSK3beta in migrating epithelial cells. J. Cell Biol 169, 929–939.[Abstract/Free Full Text]

Yang, M., Zhong, W. W., Srivastava, N., Slavin, A., Yang, J., Hoey, T., An, S. (2005). G protein-coupled lysophosphatidic acid receptors stimulate proliferation of colon cancer cells through the -catenin pathway. Proc. Natl. Acad. Sci. USA 102, 6027–6032.[Abstract/Free Full Text]

Yoshimura, T., Kawano, Y., Arimura, N., Kawabata, S., Kikuchi, A., Kaibuchi, K. (2005). GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120, 137–149.[CrossRef][Medline]

Zigmond, S. H. (2004). Formin-induced nucleation of actin filaments. Curr. Opin. Cell Biol 16, 99–105.[CrossRef][Medline]

Zumbrunn, J., Kinoshita, K., Hyman, A. A., Nathke, I. S. (2001). Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation. Curr. Biol 11, 44–49.[CrossRef][Medline]

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