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 β-spectrin on Ser/Thr is well recognized. Less clear is whether α-spectrin is phosphorylatedin vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, αII spectrin undergoesin vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified αII spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between αII spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of αII spectrin (but not the Src homology 3 domain of αI 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 αII spectrin is resistant to maitotoxin-induced cleavage by μ-calpain in vivo. In vitro studies of recombinant αII spectrin peptides representing repeats 9–12 identify two sites of pTyr modification. The first site is at Tyr¹⁰⁷³, a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr¹¹⁷⁶. This residue flanks the major site of cleavage by the calcium-dependent protease calpain, and phosphorylation of Tyr¹¹⁷⁶ by c-Src reduces the susceptibility of αII spectrin to cleavage by μ-calpain. Calpain cleavage of spectrin, activated by Ca²⁺ 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 αII spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca²⁺-mediated signaling cascades.