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.
Highlights
IntroductionOne is at the site of calpain cleavage, the other flanks a short sequence encoded by alternative exon utilization
While we cannot exclude the presence of trace phosphorylated on tyrosine (pTyr) in II spectrin that is present in these precipitates, the preponderance of pTyr-modified spectrin, identified in multiple experiments (n ϭ 6), appears to be the ␣II subunit based on the size of the pTyr immunoreactive band at Ϸ284 kDa
Tyrosine phosphorylation of ␣II spectrin was verified by immunoprecipitation with anti-pTyr followed by Western blotting (Fig. 1A, right panel)
Summary
One is at the site of calpain cleavage, the other flanks a short sequence encoded by alternative exon utilization Both sites are adjacent to the SH3 domain of spectrin, a locus that we show binds to c-Src in MDCK cell extracts. These data together with a recent independent report that appeared after this study was first submitted [14] establish a role for tyrosine phosphorylation of ␣II spectrin as a modifier of the calpain sensitivity of spectrin and reveal the spectrin cortical skeleton as a point of convergence between tyrosine kinase/phosphatase and Ca2ϩ-mediated signal transduction pathways
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