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Vol. 20, Issue 1, 56-67, January 1, 2009

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Phosphorylation of a Novel Site on the β4 Integrin at the Trailing Edge of Migrating Cells Promotes Hemidesmosome Disassembly

Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115

Submitted June 25, 2008; Revised October 27, 2008; Accepted October 30, 2008
Monitoring Editor: Richard O. Hynes

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelial cells to the basement membrane. HD components include the 6β4 integrin, plectin, and BPAGs (bullous pemphigoid antigens). HD disassembly in keratinocytes is necessary for cells to migrate and can be induced by EGF through β4 integrin phosphorylation. We have identified a novel phosphorylation site on the β4 integrin: S₁₄₂₄. Preventing phosphorylation by mutating SA₁₄₂₄ results in increased incorporation of β4 into HDs and resistance to EGF-induced disassembly. In contrast, mutating SD₁₄₂₄ (mimicking phosphorylation) partially mobilizes β4 from HDs and potentiates the disassembly effects of other phosphorylation sites. In contrast to previously described sites that are phosphorylated upon growth factor stimulation, S₁₄₂₄ already exhibits high constitutive phosphorylation, suggesting additional functions. Constitutive phosphorylation of S₁₄₂₄ is distinctively enriched at the trailing edge of migrating keratinocytes where HDs are disassembled. Although most of this S₁₄₂₄-phosphorylated β4 is found dissociated from HDs, a substantial amount can be associated with HDs near the cell margins, colocalizing with plectin but always excluding BPAGs, suggesting that phospho-S₁₄₂₄ might be a mechanism to dissociate β4 from BPAGs. S₁₄₂₄ phosphorylation is PKC dependent. These data suggest an important role for S₁₄₂₄ in the gradual disassembly of HDs induced by cell retraction.

⁺ Address correspondence to: Isaac Rabinovitz (irabinov{at}bidmc.harvard.edu)

Abbreviations used: BPAG, bullous pemphigoid antigen; EGF, epidermal growth factor; HD, hemidesmosome; OKA, okadaic acid; PKC, protein kinase C; pp, phosphopeptide; TLC, thin-layer chromatography; TLE, thin-layer electrophoresis.