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Vol. 17, Issue 10, 4330-4342, October 2006

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Tyrosine Phosphatases and Perform Specific and Overlapping Functions in Regulation of Voltage-gated Potassium Channels in Schwann Cells

Zohar Tiran^*, Asher Peretz, Tal Sines^*, Vera Shinder, Jan Sap^,||, Bernard Attali, and Ari Elson^*

Departments of *Molecular Genetics and Chemical Research Support, The Weizmann Institute of Science, Rehovot 76100, Israel; Department of Physiology and Pharmacology, Tel Aviv University Medical School, Tel Aviv 69978, Israel; and Department of Pharmacology, New York University Medical School, New York, NY 10016

Submitted February 24, 2006; Revised June 20, 2006; Accepted July 17, 2006
Monitoring Editor: Carl-Henrik Heldin

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tyrosine phosphatases (PTPs) and are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTP and PTP and the mechanisms by which they regulate K⁺ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTP inhibits Kv channels more strongly than PTP; this correlates with constitutive association of PTP with Kv2.1, driven by membranal localization of PTP. PTP, but not PTP, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTP and PTP differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reversible phosphorylation of tyrosine residues in proteins is a major mechanism for regulation of protein structure and function. Phosphorylation is regulated by the opposing activities of two superfamilies of enzymes—the protein tyrosine kinases (PTKs) and the protein tyrosine phosphatases (PTPs). More than 100 PTP genes are known in higher organisms, of which 38 belong to the classical, tyrosine-specific PTP family (Alonso et al., 2004; Andersen et al., 2004). The number of tyrosine phosphorylation sites in target proteins is significantly larger than the numbers of known PTPs or PTKs (Manning et al., 2002; Alonso et al., 2004). Each enzyme therefore interacts with several substrates, and a given substrate may be targeted by more than one kinase or phosphatase. Consequently, it is not always clear whether these enzymes fulfill molecular and physiological roles that are entirely independent or that overlap at least in part.

Molecular and physiological studies of specific PTPs in recent years indicate that the issue of functional independence versus redundancy is complex. In many cases, specific substrates are currently known to be targeted by an individual PTP; in other cases, however, substrate sharing clearly occurs. A prime example of this is Src, which can be activated by dephosphorylation of its C-terminal inhibitory tyrosine by several PTPs, including PTP1B, SHP1, PTP, and PTP (Somani et al., 1997; Ponniah et al., 1999; Su et al., 1999; Bjorge et al., 2000; Gil-Henn and Elson, 2003; Pallen, 2003). Yet, targeting of a specific substrate by several PTPs may not necessarily imply that these PTPs are fully redundant in vivo, because their expression patterns, their physical access to the substrate, and regulation of their catalytic activities may vary.

Similar complexity is found at the physiological level when phenotypes of mice genetically lacking PTPs are analyzed. Several possible modes of functional interaction between pairs of PTPs may exist. Both PTPs may have physiological functions that are either partially or entirely overlapping, in which case single mutants may have relatively weak phenotypes due to functional compensation by the other PTP. Deletion of both PTPs might then exacerbate existing phenotypes or uncover novel phenotypes. We note that the developmental phenotypes of several single-gene PTP mutants, including some of the most informative ones, are in fact rather weak and do not lead to major histopathological abnormalities in mice (e.g., Schaapveld et al., 1997; Elchebly et al., 1999; Ponniah et al., 1999; Su et al., 1999; Klaman et al., 2000; Peretz et al., 2000). In other cases, both PTPs may antagonize each other's function, evident as phenotypes of reduced intensity in mice lacking both PTPs compared with mice lacking either PTP. Along these lines, simultaneous absence of CD45 and SHP-1 corrects several B-cell defects present in mice lacking either of these PTPs (Pani et al., 1997). Antagonistic interactions have been demonstrated also in axon guidance between several Drosophila PTPs (Desai et al., 1997; Schindelholz et al., 2001; Sun et al., 2001) and between PTPRO versus RPTP and RPTP in regulating nerve structure in chick lumbar spinal cord (Stepanek et al., 2005). Last, PTPs may have separate and nonoverlapping physiological functions, in which case the phenotype of mice lacking both PTPs would seem to be a simple combination of the two single-knockout phenotypes. We note that functional redundancy or antagonism between PTPs does not necessarily imply similar interrelationships at the molecular level.

We have chosen to examine this issue by comparing the abilities of two closely related PTPs, PTP and PTP, to regulate delayed rectifier voltage-gated potassium (Kv) channels in Schwann cells. The two major forms of PTP protein are the receptor-type form (RPTP) and the nonreceptor form (cyt-PTP), which are produced by distinct promoters of the Ptpre gene (Elson and Leder, 1995a, b; Nakamura et al., 1996; Tanuma et al., 1999). Other protein forms of PTP are p67 PTP and p65 PTP, whose production is regulated at the levels of transcription and posttranslational processing, respectively (Gil-Henn et al., 2000, 2001). RPTP and cyt-PTP differ only at their amino termini, resulting in each having a distinct pattern of subcellular localization and rendering them physiologically nonequivalent. Demonstrated roles of RPTP include assisting Neu-induced mouse mammary tumor cells maintain their transformed phenotype by dephosphorylating and activating Src in vivo (Gil-Henn and Elson, 2003), and possibly down-regulating insulin receptor signaling (Moller et al., 1995; Andersen et al., 2001; Lacasa et al., 2005; Nakagawa et al., 2005). The cyt-PTP form is important for proper adhesion of osteoclasts to bone and for subsequent bone resorption (Chiusaroli et al., 2004). cyt-PTP can also down-regulate signaling mediated by the mitogen-activated protein kinase (Wabakken et al., 2002; Toledano-Katchalski et al., 2003) and by the Janus tyrosine kinase-signal transducer and activator of transcription (Tanuma et al., 2000, 2001, 2003) pathways and is required for proper functioning of mouse macrophages (Sully et al., 2001).

cyt-PTP can also down-regulate the activity of Kv channels in Schwann cells (Peretz et al., 2000). Kv channels comprise a large and ubiquitous family and are key regulators of cellular functions such as action potential waveforms, neuronal firing patterns, synaptic integration, neurotransmitter release, volume regulation, and cell proliferation. Kv channels are comprised of tetramers of membrane-spanning subunits, which may associate with up to four cytosolic regulatory subunits (Pongs, 1995; Martens et al., 1999; Yi et al., 2001). Kv channels are activated by membrane depolarization, but their activity can be modulated after tyrosine phosphorylation of their subunits by Src family PTKs (Fadool et al., 1997; Sobko et al., 1998a; Levitan, 1999; Peretz et al., 1999; MacFarlane and Sontheimer, 2000). We have shown that the subunit Kv2.1 is a physiological substrate of cyt-PTP. Kv2.1 is hyperphosphorylated in Schwann cells and sciatic nerve tissue of PTP-deficient mice; phosphorylation of Kv2.1 by Src or Fyn up-regulates channel activity, whereas channel dephosphorylation by cyt-PTP counters this effect (Peretz et al., 2000). The major site within Kv2.1 at which Src and cyt-PTP exert their opposing effects is Y124 in the cytoplasmic N terminus of the channel (Tiran et al., 2003). A stable complex between Kv2.1 and the substrate-trapping mutant D245A cyt-PTP can be isolated readily; a significant part of the stability of this complex is linked to the presence of the Y124 phosphorylation site in Kv2.1 (Peretz et al., 2000; Tiran et al., 2003). Increased Kv channel activity in Schwann cells of mice lacking PTP is correlated with transiently reduced myelination of sciatic nerve axons of these mice (Peretz et al., 2000).

The PTP most closely related to PTP is RPTP, whose major protein form is a receptor-type molecule (Krueger et al., 1990). RPTP activates Src by dephosphorylating its C-terminal inhibitory tyrosine residue in a manner similar to PTP (for review, see Pallen, 2003). Activation of Src and related kinases by RPTP mediates many of its physiological functions, such as regulation of integrin signaling and cell migration (Ponniah et al., 1999; Su et al., 1999; von Wichert et al., 2003; Zeng et al., 2003) and neurite elongation (Bodrikov et al., 2005). RPTP has been suggested to down-regulate insulin receptor signaling (Moller et al., 1995; Lammers et al., 1997; Andersen et al., 2001; Lacasa et al., 2005), although lack of RPTP does not seem to affect glucose homeostasis in vivo (Le et al., 2004). At the whole-animal level, gene-targeting studies have established that RPTP functions in developmental positioning of hippocampal pyramidal neurons, learning, and neuroplasticity (Petrone et al., 2003; Skelton et al., 2003). Molecular targets of RPTP responsible for these phenotypes remain to be identified. Interestingly, ectopically expressed RPTP is capable of activating the Kv1.2 and Kv1.1 potassium channels upon signaling by the m1 muscarinic and serotonin receptors (Tsai et al., 1999; Imbrici et al., 2000). The difference between this effect and inhibition of Kv2.1 by cyt-PTP may stem from differences in the biophysical attributes of the two channel subtypes, the biochemical properties of either PTP, or from differences in the cellular context or specific signaling pathways involved in either system.

A significant part of the above-mentioned information has been obtained by use of mice genetically lacking either PTP or PTP (Ponniah et al., 1999; Su et al., 1999; Peretz et al., 2000; Sully et al., 2001; Petrone et al., 2003; Bodrikov et al., 2005). Yet, lack of either PTP is tolerated well by mice and is not associated with major morbidity or mortality. Although PTPs and are products of separate genes and have distinct expression patterns in vivo, these PTPs are highly related. PTPs and are the only two members of the type IV family of receptor PTPs (Alonso et al., 2004; Andersen et al., 2004), and 83% of the amino acid residues in their cytosolic domains are either identical or similar, suggesting that functional redundancies may exist between them. To examine this issue in vivo, we derived and examined mice lacking both PTP and PTP. In this study, we examine regulation of Kv channel activity and Src in Schwann cells in the absence of either or both PTPs and conclude that some of the functions of these closely related PTPs are clearly divergent within a single cell type.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmids
Full-length mouse cDNAs for cyt-PTP (Elson and Leder, 1995a), RPTP (Elson and Leder, 1995b), and RPTP (kind gift of the late Dr. M. Thomas) were used in this study. Lck-cyt-PTP was prepared by polymerase chain reaction (PCR)-mediated addition of the N-terminal Lck myristoylation domain to the N terminus of cyt-PTP; cyt-PTP was prepared similarly by replacing the extracellular and transmembranal domains of RPTP (amino acids 1–166) with the 12 N-terminal residues of cyt-PTP. The following mutants were prepared by site-directed mutagenesis: R340M RPTP, R283M cyt-PTP, D245A cyt-PTP, D437A RPTP, D245A Lck-cyt-PTP, and D437A cyt-PTP. Numbering of the mutated residue in the latter two cDNAs is unchanged from cyt-PTP and RPTP, respectively, for clarity. All products of PCR and of site-directed mutagenesis were sequenced before use, and all contained a C-terminal FLAG tag. Also used were the cDNAs for rat Kv2.1 (gift of Drs. J. Barhanin and M. Lazdunski), Y124F Kv2.1 (Tiran et al., 2003), and chicken Y527F Src (gift of Dr. S. Courtneidge, Van Andel Research Institute, Grand Rapids, MI). All cDNAs were cloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA).

Generation and Analysis of Mice
Mice were housed (up to 5 per cage) in the Weizmann Institute barrier mouse facility. Animals were handled in accordance with National Research Council regulations, Israeli Law, and Weizmann Institute regulations; all studies were approved by the Institutional Animal Care and Use Committee. Mice lacking PTP (Peretz et al., 2000) or PTP (Su et al., 1999) were crossed; progeny were interbred to generate the four genotypes used in this study—wild type (WT), PTP-deficient (EKO or Ptpre–/–), PTP-deficient (AKO or Ptpra–/–), and deficient in both PTPs (DKO or Ptpre–/–/Ptpra–/–)—in the same genetic background (C57Bl/6 x 129Sv/Ev).

Genotyping of mice by PCR for the PTP-targeted allele was done using primers WT5' (5'-ACTCCCAGACAGCTGCAAAGC-3') and WT3' (5'-CGCTACAGTGAACCACAATGG-3'), which amplify a 250-base pair fragment from the WT allele of PTP, and primers SL3015' (5'-GGATCCAATTGCAATGATCA-3') and NEOKO3' (5'-ACTGAAGGCTCTTTACTATTGC-3'), which amplify a 450-base pair fragment from the targeted PTP allele. Reactions included 1-µl tail biopsy DNA, 30 pmol of each oligonucleotide, 0.25 mM of each dNTP (Sigma-Aldrich, St. Louis, MO), 1x polymerase reaction buffer [1.5 mM MgCl₂, 20 mM (NH₄)SO₄, 75 mM Tris-HCl, pH 9.0, and 0.01% Tween 20], and 1.25 U of Taq polymerase (JMR Holdings, London, United Kingdom) in a final volume of 25 µl. Samples were denatured at 93°C for 2 min followed by 30 cycles of 93°C for 30 s, 52°C for 1 min, and 72°C for 1 min. Genotyping by PCR for the RPTP-targeted allele was performed as described previously (Su et al., 1999). Mice were weaned at 3 wk of age and were weighed once a week starting 40 d after birth through adulthood (5–7 mo). The following tissues were stained with hematoxylin and eosin and examined by light microscopy: liver, stomach, small and large intestines, kidney, adipose connective tissue, uterus, skeletal muscle (diaphragm), various peripheral nerves, pancreas, adrenal, brown fat, various blood vessels, heart, thymus, spleen, lung, lymph nodes (mesenteric, peripheral, and inguinal), skull (coronal sections), skin (head), marrow (sternum), thyroid, salivary glands, tongue, pancreas, oviducts, urinary bladder, and brain. No gross abnormalities beyond those described for AKO mice (Su et al., 1999) were detected.

Electron Microscopy
Sciatic nerves from 5-d-old mice (6–11 mice from each genotype from at least 2 different litters born to different parents) were analyzed as described previously (Peretz et al., 2000). Samples from different mice were always taken from the same location, 5 mm from the proximal end of the nerve. Briefly, samples were fixed with 3% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 1% OsO₄ and 2% uranyl acetate, dehydrated in a graded series of ethanols, and embedded in Epon-812. Transverse cross-sections of 70–90 nm thickness were cut with a Leica Ultracut UCT ultramicrotome (Leica, Wetzlar, Germany), stained with uranyl acetate and lead citrate, and examined on a Tecnai 12 transmission electron microscope at 120Kv. Cross-sections were examined from each mouse at 4000x magnification. Pictures were digitized with a MegaView III charge-coupled device camera and analyzed with AnalySIS software (SPSS, Chicago, IL). The thickness of the myelin sheath of all axon profiles in a given field was measured, and data for mice of the same genotype was pooled.

Cell Culture
Primary Schwann cells were prepared from sciatic nerves of 3- to 5-d-old pups as described previously (Sobko et al., 1998a). Age-matched WT, EKO, AKO, or DKO pups were obtained from separate matings of homozygous mice of the same genotypes. Schwann cells were grown in DMEM/F-12 (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Invitrogen), 2 mM glutamine, and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) and analyzed 8–10 d after isolation. Human embryonic kidney (HEK)293 cells were grown in DMEM (Invitrogen) supplemented with 10% FCS and antibiotics as described above and transfected by the calcium phosphate technique.

Electrophysiology
Whole-cell currents were recorded from Schwann cells by using the patch-clamp technique as described previously (Sobko et al., 1998a). Signals were amplified using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA), sampled at 2 kHz, and filtered below 0.8 kHz via a four-pole Bessel low-pass filter. Data acquisition was done using the pClamp 8.1 software (Molecular Devices). The patch pipettes were filled with 164 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 11 mM EGTA, 10 mM HEPES, and 11 mM glucose at pH 7.4 and had a tip resistance of 4–8 M. The external solution contained 140 mM NaCl, 5 mM KCl, 5 mM CaCl₂, 2 mM MgCl₂, 11 mM glucose, and 10 mM HEPES at pH 7.4. Series resistances (3–13 M) were compensated (75–90%) and periodically monitored.

Single-channel recordings were performed in Schwann cells using the cell-attached configuration of the patch-clamp technique. Cells were continuously perfused with a standard salt solution containing 150 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1.2 mM MgCl₂, 15 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). Pipettes were pulled from borosilicate glass, coated with Sylgard, and filled with the above-mentioned standard salt solution (8–10 M). Signals were sampled at 10 kHz and low-pass filtered at 2 kHz. Data were analyzed using Clampfit 9.2 (Molecular Devices).

Biochemical Fractionation of Sciatic Nerve Tissue and Cells
For analysis of Kv2.1 phosphorylation in sciatic nerves, nerve tissue was dissected from 3-d-old mice (30–40 mice per experimental point) and stored in liquid nitrogen until use. Tissue was homogenized in buffer containing 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM sodium fluoride, 0.5 mM sodium pervanadate, and protease inhibitors [1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 µM bestatin, 15 µM E-64, 20 µM leupeptin, and 15 µM pepstatin; Sigma-Aldrich], and centrifuged at 21,000 x g for 30 min at 4°C. The pellet (crude membranal fraction) was resuspended and sonicated in solubilization buffer (10% glycerol, 50 mM HEPES, pH 7.4, 10 mM EDTA, 150 mM NaCl, 1.5 mM MgCl₂, 1% Triton X-100, 1 mM PMSF, 50 mM sodium fluoride, 0.5 mM sodium pervanadate, and protease inhibitors). The sonicate was incubated with shaking in solubilization buffer for 1 h at 4°C, spun at 21,000 x g, and the supernatant was collected. Membranal extracts (1.5–3 mg) were incubated with shaking with anti-Kv2.1 monoclonal antibodies (Trimmer, 1991) at 4°C overnight. Immune complexes were collected by adding protein A/G-Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA) and incubating in solubilization buffer for 2 h at 4°C with shaking, followed by three washes in solubilization buffer. In other studies, cultured cells were fractionated into membranal and nonmembranal (i.e., combined cytosolic and nuclear) fractions by homogenization in hypotonic buffer as described previously (Elson and Leder, 1995a).

Immunoprecipitation and Protein Blotting
Cells were lysed in buffer A (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, and 1% NP-40), supplemented with protease inhibitors and either 0.5 mM sodium pervanadate (for phosphorylation studies) or 5 mM sodium iodoacetate (for substrate-trapping studies). In phosphorylation studies crude cellular proteins (1 mg) were reacted with anti-phosphotyrosine antibodies (clone PY20; BD Biosciences Transduction Laboratories, Lexington, KY) and protein A-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) for 2 h at 4°C, spun down, and washed three times in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS). In substrate-trapping studies cellular proteins (1 mg) were reacted with anti-FLAG M2 affinity beads (Sigma-Aldrich) for 4–6 h, followed by two washes in buffer A and one wash in RIPA buffer. SDS gel electrophoresis, blotting, and antibody hybridization were done as described previously (Gil-Henn et al., 2000). Primary antibodies used included rabbit polyclonal anti-PTP, which cross reacts with RPTP (Elson and Leder, 1995b), monoclonal [clone D4/11, generous gift of Drs. E. Peles (Weizmann Institute of Science, Rehovot, Israel) and J. Trimmer (University of California, Davis, CA) (Trimmer, 1991)] or polyclonal (Alomone Labs, Jerusalem, Israel) anti-Kv2.1, monoclonal anti-v-Src (clone 327; Calbiochem, San Diego, CA), and monoclonal anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY). Band signals at nonsaturating exposures were scanned and measured by image densitometry by using Image Gauge 4.0 (FujiFilm, Tokyo, Japan) and Adobe Photoshop 7.0 software (Adobe Systems, Mountain View, CA).

Src Activity Assay
Sciatic nerve tissue was dissected from 3- to 5-d-old mice and stored frozen in liquid nitrogen until used. Tissue was homogenized in buffer A supplemented with protease inhibitors and sodium pervanadate as described above, sonicated, and spun to remove debris. Crude lysates (0.3–0.5 mg) were used to determine activity of immune-precipitated c-Src by using [-³²P]ATP and enolase as described previously (Gil-Henn and Elson, 2003).

In Vitro Dephosphorylation of pKv2.1
HEK293 cells expressing FLAG-tagged RPTP or cyt-PTP were lysed in buffer A supplemented with protease inhibitors but without pervanadate. PTPs were immunoprecipitated with monoclonal anti-FLAG M2 antibodies (Sigma-Aldrich). Precipitated material was washed three times in buffer A, twice in buffer B (20 mM HEPES, pH 7.6, 100 mM KCl, 0.5 mM EDTA, 0.4% NP-40, and 20% glycerol), followed by two short (1.5-min) washes in buffer 54K (50 mM Tris, pH 7.9, 150 mM NaCl, and 0.5% Triton X-100). Proteins were eluted by two incubations in equal volumes of Tris-buffered saline buffer (20 mM Tris-Cl, pH 7.35, and 150 mM NaCl) containing 1 mg/ml FLAG peptide (Sigma-Aldrich) and 0.1 mM EGTA, at 32°C for 3 min. Eluted material was pooled. FLAG-tagged phosphorylated Kv2.1 was purified in a similar manner from HEK293 cells expressing Kv2.1 and active (Y527F) Src, except that the lysis buffer A contained 0.5 mM sodium pervanadate. Protein purity and amounts were determined by gel electrophoresis and silver staining. pKv2.1 (15 ng) was incubated either alone or with 100 ng of purified PTP in PTP activity buffer [50 mM 2-(N-morpholino)ethanesulfonic acid, pH 7.0, 0.5 mg/ml bovine serum albumin, and 0.5 mM dithiothreitol] at 32°C for 6 h, followed by adding SDS-PAGE sample buffer and boiling. Phosphorylation of Kv2.1 was analyzed by SDS-PAGE and protein blotting with anti-pTyr antibodies as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice Lacking Both PTP and PTP Are Viable
The effects of absence of PTP or PTP on Kv channels were compared in mice lacking either PTP (EKO mice; Peretz et al., 2000) or PTP (AKO mice; Su et al., 1999). To obtain additional information on the functional interactions between these PTPs, EKO and AKO mice were crossed to generate mice lacking both PTPs (DKO mice). DKO mice were viable and produced litters of normal size. Analysis of protein expression from various tissues confirmed that DKO mice lacked RPTP and the various PTP proteins (Figures 1A and 3A). Interestingly, DKO mice of both genders weighed 15–20% less than WT, AKO, or EKO mice (Figure 1B). Reduced DKO body weight was detected before weaning and persisted throughout adulthood, indicating that DKO mice gain weight proportionally at the same rate as WT mice and consequently remain smaller throughout life. This result indicates that PTP and PTP perform a redundant role in regulation of mouse body weight. Altered body weight is a complex phenotype that has been observed in mice lacking various unrelated genes, among them PTPs (Klaman et al., 2000; Cheng et al., 2002; Zhang et al., 2004). Further studies will therefore be needed to uncover its cellular and molecular bases in this particular case. Histological examination of several tissues of DKO mice did not reveal major gross structural abnormalities beyond those described for AKO mice (Su et al., 1999; Petrone et al., 2003); the tissues examined are listed in Materials and Methods. We conclude that abnormalities in these tissues, if they exist, are subtle; their detection may require use of more sensitive techniques, as shown below in the case of sciatic nerves.

View larger version (39K): [in this window] [in a new window]   Figure 1. PTP expression and body weight of mice used in this study. (A) Expression of RPTP and RPTP in brain lysates. Blots containing crude extracts of brain from adult WT, AKO, EKO, and DKO mice were reacted with anti-PTP antibodies, which also cross-react with RPTP, to assess expression of PTP and PTP in this tissue. Asterisks mark nonspecific bands; molecular mass standards are in kilodaltons. (B) Weights of mice of the indicated genotypes are shown between 40 and 150 d of age. The weight of DKO mice was significantly (15–25%) lower than that of WT, AKO, or EKO mice (Mann–Whitney U-test, p < 0.0001). EKO males weighed slightly less than WT males up to 90 d (Mann–Whitney U-test, p < 0.01). Each data point represents the average weight ± SEM with n = 20–51. Asterisks mark data points significantly different from WT. Similar results were obtained with female mice (our unpublished data).

View larger version (71K): [in this window] [in a new window]   Figure 2. Reduced myelination of axons in sciatic nerves of 5-d-old AKO, EKO, and DKO mice. (A) Distribution of thicknesses of myelin sheaths in wild-type, AKO, EKO, and DKO sciatic nerves (WT, A, E, and D, respectively). n = 1370–2320 individual axons per genotype, as indicated below. (B) Bar diagram comparing the average sheath thickness (in nanometers, ± SEM). WT, 284.2 ± 3.0, n = 2320 sheaths; EKO, 232.6 ± 3.3, n = 1652; AKO, 251.7 ± 3.8, n = 1370; and DKO, 214.9 ± 5.4, n = 1601. All knockout means are distinct from WT mean (p < 0.0001); EKO and AKO means are distinct from DKO mean (EKO, *p = 0.0054); AKO, **p < 0.0001). Statistical significance was determined by Welch's t test. (C) Representative electron microscope pictures of transverse cross-sections through sciatic nerves of 5 d-old WT, AKO, EKO, and DKO mice. Arrows mark several cross-sections of myelinated axons, evident as closed dark figures. Arrowheads mark several unmyelinated axons.

Increased Activation of Kv Channels in Schwann Cells of AKO Mice
Previous analyses revealed that Kv channel activity was increased in primary Schwann cells of EKO mice (Peretz et al., 2000). To examine the effect of lack of PTP on regulation of Kv channels, we examined the activity of Kv channels in Schwann cells of AKO mice. Primary Schwann cells express both RPTP and the nonreceptor form of PTP, cyt-PTP; as expected, either PTP was absent from Schwann cells of the corresponding single knockout mice (Figure 3A). Primary Schwann cell cultures from 3- to 5-d-old mice of the various genotypes were prepared and used for recording voltage-gated potassium currents by using the whole-cell patch-clamp technique. Schwann cells of EKO and of AKO mice exhibited a marked increase in K⁺ current densities (Figure 3, B and C), with the effect in AKO mice much stronger than in EKO mice (47% increase in EKO Schwann cells compared with WT (at +60 mV), versus an increase of 150% in AKO Schwann cells). This result indicates that RPTP and cyt-PTP each inhibit Kv channel activity in Schwann cells, but they do so most likely by distinct mechanisms. To determine the effects of simultaneous inactivation of both PTPs on current density we analyzed Schwann cells from DKO mice, which are devoid of both cyt-PTP and RPTP proteins (Figure 3A). Kv channel activity in DKO Schwann cells was lower than in AKO cells and was slightly higher than in EKO cells (62% increase compared with WT (Figure 3, B and C). Thus, absence of both PTPs did not increase Kv currents in an additive manner, possibly suggesting that cyt-PTP may also promote Kv channel activity in the absence of RPTP as discussed further below.

View larger version (26K): [in this window] [in a new window]   Figure 3. Increased Kv channel currents in primary Schwann cells of EKO, AKO, or DKO mice. (A) Blots containing crude extracts from Schwann cells from 3- to 5-d-old WT, AKO, EKO, and DKO mice were reacted with anti-PTP antibodies to assess expression of cyt-PTP and PTP. Asterisks mark nonspecific bands; molecular mass markers are in kilodaltons. (B) The K+ current density (picoamperes/picofarads; mean ± SEM) of WT (n = 12), AKO (n = 11), EKO (n = 14), and DKO (n = 9) Schwann cells plotted against voltage steps (millivolts). At +60 mV, EKO cells exhibit a 47% increase in current density compared with WT cells; analogous values for AKO and DKO cells are +150 and +62%, respectively. (C) Whole-cell K+ currents recorded from primary Schwann cells of the four genotypes. Cells were stepped from a holding potential of –80 to +60 mV in + 10-mV increments for a 400-ms pulse duration.

View larger version (21K): [in this window] [in a new window]   Figure 4. Single-channel properties of Schwann cell K+ currents measured in cell-attached patches. (A) Single channel current traces of WT and EKO Schwann cells are displayed at two different potentials evoked from the cell resting potential (–50 mV). The closed channel state is indicated by the dashed lines. The K+ concentration in the pipette and the bath was 2.5 mM. (B) Representative unitary current–voltage relations measured from WT and EKO Schwann cells (of 21 similar cells). The straight lines correspond to the fit of data points and indicate unitary conductance of 10 pS. (C) Open probability (mean ± SEM) determined from the analysis of 17–21 cells of each the four genotypes. Differences between the four genotypes are not statistically significant.

Hyperphosphorylation of Kv2.1 and Deregulation of c-Src in the Absence of Either or Both PTP and PTP

View larger version (26K): [in this window] [in a new window]   Figure 5. Phosphorylation of Kv2.1 and Src activity in sciatic nerves of WT, EKO, AKO, and DKO mice. (A) Crude membrane fractions of sciatic nerves from 3-d-old WT, EKO, AKO, and DKO mice were prepared and solubilized. After immunoprecipitation with anti-Kv2.1 antibodies, precipitates were blotted with anti-pTyr antibodies (top) followed by stripping and blotting with anti-Kv2.1 antibodies (bottom). Blots are from a representative experiment of four performed. (B) Bar diagram showing Kv2.1 phosphorylation intensity (± SEM) normalized to Kv2.1 protein content and presented relative to WT (= 100): EKO = 320.4 ± 105.8, AKO = 216.0 ± 58.3, and DKO = 231.8 ± 57.4; n = 4 repeats. (C) Src protein was immunoprecipitated from sciatic nerve extracts of 3- to 4-d-old mice and allowed to catalyze incorporation of 32P into enolase. Reaction mixture was subject to SDS-PAGE and blotting. Blot was exposed to document enolase-associated radioactivity (top) and then probed with anti-Src antibody to document amount of Src protein present in the precipitate (bottom). (D) Bar diagram showing relative Src activity (± SEM) in the four genotypes studied: WT = 1.00 ± 0.04; EKO = 0.89 ± 0.09; AKO = 0.50 ± 0.02; and DKO = 0.61 ± 0.05. n = 3–5 experiments per genotype. AKO and DKO are statistically different (asterisks) by Student's t test from WT (p 0.0006) and EKO (p 0.018). WT versus EKO and AKO versus DKO are not statistically distinct.

Kv2.1 Interacts Constitutively with RPTP, but Not with cyt-PTP
The above-mentioned results suggest that RPTP, but not cyt-PTP, may regulate Kv channels in Schwann cells also indirectly by affecting Src activity. However, this parameter is not sufficient to explain in full molecular detail the stronger inhibition of Kv channel activity by RPTP. To further address this issue, we examined the abilities of either PTP to interact with and to dephosphorylate Kv channels.

Experiments using isolated cyt-PTP, RPTP, and Src-phosphorylated Kv2.1 indicated that either PTP can dephosphorylate Kv2.1 in vitro (Figure 6A). However, their respective activities toward Kv2.1 may differ in a cellular context. RPTP may have better access to Kv channel proteins because it is an integral membrane protein; this is in contrast to cyt-PTP, which is predominantly cytosolic (Elson and Leder, 1995a). Alternatively, the membrane-spanning and extracellular domains of RPTP, which have no counterparts in cyt-PTP, might interact with Kv2.1 and contribute to its regulation. Last, slight differences between the sequences of the active sites and regulatory regions of these highly related PTPs may make RPTP a more effective down-regulator of Kv2.1.

View larger version (28K): [in this window] [in a new window]   Figure 6. PTP and PTP regulate tyrosine phosphorylation of Kv2.1. (A) RPTP and cyt-PTP can dephosphorylate tyrosine-phosphorylated Kv2.1 in vitro. Top, phosphorylated Kv2.1 remaining after incubation without added PTP or after addition of purified cyt-PTP or RPTP as indicated. Remaining panels document addition of purified pKv2.1, cyt-PTP, and RPTP to the various reactions. FL, full-length cyt-PTP. Anti-PTP antibodies cross-react with RPTP. (B) RPTP and cyt-PTP reduce Src-mediated phosphorylation of Kv2.1 in cells. HEK293 cells stably expressing Kv2.1 were transiently transfected with activated (Y527F) Src, together with RPTP, cyt-PTP, RPTP, or the inactive mutants R340M RPTP and R283M cyt-PTP. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies followed by blotting with anti-Kv2.1 antibodies. Blots containing crude extracts were reacted with anti-Kv2.1, anti-PTP, and anti-Src antibodies to assess expression of these proteins. Blots are from a representative experiment of three performed. (C) Constitutive association of Kv2.1 with RPTP. HEK293 cells were transiently transfected with Kv2.1 together with FLAG-tagged cyt-PTP, RPTP, or their substrate-trapping mutants (D245A cyt-PTP and D437A RPTP), as indicated. Following cell lysis and anti-FLAG immunoprecipitation, coprecipitating Kv2.1 was detected by blotting with anti-Kv2.1 antibodies (top). Second and third panels depict amounts of precipitating PTPs and expression of Kv2.1 in the crude lysates, respectively. Asterisks denote p66 PTP, a cytosolic cleaved form of RPTP (Gil-Henn et al., 2001).

We next compared the ability of substrate-trapping mutants of cyt-PTP and RPTP to bind WT Kv2.1. Mutants of this type (D245A cyt-PTP and D437A RPTP) are almost entirely catalytically inactive, but their catalytic sites can still bind phosphorylated substrates (Flint et al., 1997; Peretz et al., 2000; Tiran et al., 2003; Blanchetot et al., 2005). Severalfold more Kv2.1 coprecipitated with the substrate-trapping D245A mutant of cyt-PTP than with WT cyt-PTP (Figure 6C), indicating that a significant part of the interactions between Kv2.1 and cyt-PTP is mediated by the active site of PTP. In contrast, similar amounts of Kv2.1 associated with the WT RPTP and with its substrate-trapping mutant D437A RPTP (Figure 6C). The interactions between Kv2.1 and RPTP are therefore more stable and are likely to involve other regions in addition to the active site, in agreement with the stronger effect of RPTP on Kv channel activity documented above.

RPTP is an integral membrane protein with membrane-spanning and extracellular domains. cyt-PTP lacks such domains; although a fraction of cyt-PTP molecules are membrane associated, most are cytosolic. To examine the effects of membrane localization on the ability to interact with Kv2.1, we turned to a series of D-to-A substrate-trapping mutants shown in Figure 7A. This series included cyt-PTP, an artificial cytoplasmic derivative of RPTP in which the transmembranal and extracellular regions of RPTP were replaced with the first 12 amino acids of cyt-PTP. These 12 residues are unique to cyt-PTP and regulate membranal localization of this isoform (Gil-Henn et al., 2000); access of cyt-PTP to the cell membrane is similar to that of cyt-PTP (Figure 7B). In a second construct (Lck-cyt-PTP), cyt-PTP was targeted to the plasma membrane by an N-terminal Lck dual myristoylation motif (Zlatkine et al., 1997). This construct is predominantly membrane-associated, but it lacks membrane-spanning and extracellular domains. The series also included RPTP, the receptor-type isoform of PTP, as well as cyt-PTP and RPTP. All molecules were expressed in HEK293 cells, and their expected subcellular localization was verified by biochemical fractionation (Figure 7B). Full-length cyt-PTP was mostly localized in the cytoplasmic fraction with a portion present in the membranal fraction, as shown previously (Elson and Leder, 1995a). Adding the Lck motif in Lck-cyt-PTP resulted in a significant shift of full-length cyt-PTP to the membrane fraction; the receptor-type RPTP was found exclusively in the membrane fraction. In all three cases, p67 and p65, which are coexpressed with the full-length PTP molecules but which lack membrane-targeting sequences, were found in the cytosol. RPTP was found exclusively at the cell membrane, and cyt-PTP was expressed at similar levels in the cytoplasmic and membranal fractions. The cellular expression patterns of these two PTP proteins are in good agreement with their PTP counterparts—RPTP and cyt-PTP, respectively.

View larger version (51K): [in this window] [in a new window]   Figure 7. Membranal localization of PTPs and drives their constitutive interaction with Kv2.1. (A) Schematic representation of the various FLAG-tagged substrate-trapping D-to-A mutants of PTP and PTP used in this study: D437A RPTP, D437A cyt-PTP, D245A cyt-PTP, D245A Lck-cyt-PTP, and D302A RPTP. The mutated aspartic residue (asterisk) is the same in all cases, although its exact numbering differs among the various molecules. Residue numbering in cyt-PTP and Lck-cyt-PTP are as in RPTP and cyt-PTP, respectively, for clarity. (B) The subcellular localization of molecules shown in A. After transfection into HEK293 cells, cells were fractionated and the cytoplasmic (C) and membranal (M) fractions were analyzed on 7% SDS-PAGE gels and blotted with anti-PTP/ antibodies. (C) Constitutive association with Kv2.1 is dependent on membrane localization. HEK293 cells were transiently transfected with WT Kv2.1 (Y) or Y124F Kv2.1 (F) together with the indicated FLAG-tagged substrate-trapping D-to-A mutants. After cell lysis and anti-FLAG immunoprecipitation, amount of coprecipitating Kv2.1 was assessed by blotting. Shown is a representative set of blots documenting coimmunoprecipitation of WT versus Y124F Kv2.1 with the various trapping mutants used. Also shown are amounts of precipitating PTP and Kv2.1 expression in the crude lysates (second and third panels, respectively). (D) Bar diagram showing Y124F Kv2.1 coimmunoprecipitated with substrate-trapping D-to-A mutants normalized to protein content. Values (mean ± SEM) are relative to association of WT Kv2.1 with each phosphatase. D437A cyt-PTP and D245A cyt-PTP exhibit reduced binding to Y124F Kv2.1 compared with WT Kv2.1 (63.3 ± 10.4 and 55.5 ± 13.8%, respectively; p = 0.03–0.05; Welch's t test). In contrast, the membrane-bound mutants D437A RPTP, D245A Lck-cyt-PTP, and D302A RPTP bind WT and Y124F Kv2.1 similarly.

Examination of binding of the various PTP substrate-trapping mutants to WT versus Y124F Kv2.1 revealed a clear distinction between receptor-type and nonreceptor-type forms of the PTPs (Figure 7D). Approximately 45% less Y124F Kv2.1 coprecipitated with D245A cyt-PTP compared with WT Kv2.1, despite similar expression of both forms of Kv2.1 and similar precipitation of D245A cyt-PTP in these cells (Figure 7C). Less Y124F Kv2.1 also coprecipitated with D437A cyt-PTP compared with WT Kv2.1; in both cases the reductions in association were statistically significant. In contrast, associations of D302A RPTP with WT and Y124F Kv2.1 were comparable; similar results were obtained using D437A RPTP (Figure 7D). Together, these results indicate strongly that the receptor-type forms of both PTPs can bind Kv2.1 constitutively, whereas their cytosolic forms do so much less efficiently; strong binding segregates with cellular localization and not with PTP identity. The differences observed between RPTP and cyt-PTP in binding Kv2.1 are therefore not the result of sequence differences between these two distinct PTPs or in the phosphotyrosine residues they target in Kv2.1, but rather they are the result of one of them being a receptor-type molecule, whereas the other is not.

A question left unresolved was whether RPTP and RPTP bind Kv2.1 constitutively because their membranal localization makes Kv2.1 more readily accessible or because their membrane-spanning and extracellular domains mediate additional interactions with Kv2.1. To resolve this issue, binding of D245A Lck-cyt-PTP to WT and to Y124F Kv2.1 was compared. This membrane-associated protein behaved similarly to the two receptor-type forms and bound similar amounts of WT and of Y124F Kv2.1, despite lacking membrane spanning and extracellular domains (Figure 7, C and D). We therefore conclude that the membrane-spanning and extracellular domains of RPTP and RPTP do not play a significant role in binding Kv2.1. Localization at the cell membrane is sufficient to drive constitutive association between PTPs and and Kv2.1. The inherent differences between the effects of either PTP on this channel protein are most likely the result of their differing patterns of subcellular localization in Schwann cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Comparison of the phenotypes of EKO and AKO mice indicates that the functional overlaps and interactions between PTPs and in Schwann cells are complex and context dependent. Analyses of sciatic nerve axons of 5-d-old mice revealed significant reductions in myelin sheath thickness in AKO, EKO, and DKO mice compared with WT mice. The effects of lack of PTP and PTP were additive, with DKO mice most severely affected. These results indicate that both PTP and PTP support sciatic nerve myelination in a manner that is at least partially nonredundant and that the time period when this role is most significant is in early postnatal life. The transient nature of the myelination phenotype implies that as mice age, the need for PTP and PTP in regulating myelination decreases or that other PTPs compensate for lack of these two PTPs. Possible candidates for such compensation include CD45 (Nakahara et al., 2005), SHP-1 (Massa et al., 2004), PTP (Harroch et al., 2002), and PTP (Wallace et al., 1999), because mice separately lacking these PTPs exhibit defects in myelination and/or in recovery of myelination after damage to neurons. Nonetheless, existence of myelination defects in postnatal EKO, AKO, and DKO mice indicates that PTP and PTP perform a unique function in Schwann cells and that other gene products cannot replace these enzymes at this developmental stage.

Previous reports have established a correlation between decreased Kv channel activation on the one hand, and exit of Schwann cells from the cell cycle and onset of myelination on the other hand (Sobko et al., 1998a; MacFarlane and Sontheimer, 2000). Increased Kv channel activity observed in Schwann cells of AKO and EKO mice can suggest that loss of cyt-PTP or RPTP reduces myelination by preventing the normal decrease in Kv channel activation that occurs as Schwann cells mature in early postnatal mice. However, this possibility does not hold up well in the more complex setting of DKO mice, where the largest decreases in myelination occur together with less-than-maximal increases in Kv channel activation. Together, our observations suggest that RPTP and cyt-PTP can affect sciatic nerve myelination by additional mechanisms beyond regulation of Kv channels. For example, a possible additional mechanism for linking PTP activation and Schwann cell-mediated myelination is cell adhesion, which is necessary for proper axonal myelination. The involvement of RPTP in cell adhesion is well established, evident in its ability to control integrin-mediated responses through activation of Src (Su et al., 1999), to form a complex with contactin (Zeng et al., 1999), to regulate integrin-stimulated focal adhesion kinase autophosphorylation and cytoskeletal rearrangement in cell spreading and migration (Zeng et al., 2003), and to participate in neural cell adhesion molecule-mediated signaling (Bodrikov et al., 2005). cyt-PTP is also involved in cell adhesion, because osteoclasts from EKO mice do not adhere well to matrix and display significantly disorganized adhesion structures (Chiusaroli et al., 2004). Further studies will indicate whether these effects participate in regulating myelination in this system. Finally, we note that axons from AKO and EKO mice are slightly thicker than their WT counterparts. Thicker axons are often more heavily myelinated; hence, reduced myelination in AKO and EKO axons is probably not caused by this change in axon diameter. Conversely, absence of both PTPs results in a slight reduction in axon diameter in DKO mice, which may possibly contribute to the reduced myelination of these axons. In all, these results indicate that absence of either or both PTPs can affect the axons themselves as well. Further studies are required to better understand this issue.

The experimental system studied here allows better understanding of the molecular mechanisms by which PTPs and regulate Kv channels. Loss of either cyt-PTP or RPTP in primary Schwann cells resulted in marked increases in maximal K⁺ current density and in increased tyrosine phosphorylation of Kv2.1. Importantly, these results indicate that the ultimate physiological effect of either PTP in Schwann cells in vivo is to inhibit phosphorylation and activation of Kv channel proteins. In principle, increased K⁺ current density may be caused by an increase in the number of functional channels available at the plasma membrane, a rise in Po, and/or elevated channel unitary conductance. Our single-channel analyses indicate that the increase in Schwann cell K⁺ current density observed in the PTP knockout mice cannot be accounted for by differences in Po or in unitary conductances and most likely results from increased numbers of functional channels available at the plasma membrane. The gross expression levels of Kv2.1 and Kv1.5 in primary Schwann cells from all four genotypes are similar (Figure 5A; our unpublished data). However, channel availability may be regulated by more subtle means such as interactions with other proteins or subcellular localization of channel molecules. The latter phenomenon has been described recently in