C-Src Binds αII Spectrin's Src Homology 3 (SH3) Domain and Blocks Calpain Susceptibility by Phosphorylating Tyr1176

Abstract

Spectrin is a ubiquitous heterodimeric scaffolding protein that stabilizes membranes and organizes protein and lipid microdomains on both the plasma membrane and intracellular organelles. Phosphorylation of beta-spectrin on Ser/Thr is well recognized. Less clear is whether alpha-spectrin is phosphorylated in vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, alphaII spectrin undergoes in vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified alphaII spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between alphaII spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of alphaII spectrin (but not the Src homology 3 domain of alphaI spectrin) bind specifically to phosphorylated c-Src in Madin-Darby canine kidney cell lysates, suggesting that this kinase is responsible for its in vivo phosphorylation. pTyr-modified alphaII spectrin is resistant to maitotoxin-induced cleavage by mu-calpain in vivo. In vitro studies of recombinant alphaII spectrin peptides representing repeats 9-12 identify two sites of pTyr modification. The first site is at Tyr(1073), a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr(1176). This residue flanks the major site of cleavage by the calcium-dependent protease calpain, and phosphorylation of Tyr(1176) by c-Src reduces the susceptibility of alphaII spectrin to cleavage by mu-calpain. Calpain cleavage of spectrin, activated by Ca(2+) and calmodulin, contributes to diverse cellular processes including synaptic remodeling, receptor-mediated endocytosis, apoptosis, and the response of the renal epithelial cell to ischemic injury. Tyrosine phosphorylation of alphaII spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca(2+)-mediated signaling cascades.