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Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes.

ABSTRACT: Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified alphaII-spectrin as such a VASP-interacting protein. alphaII-Spectrin binds to the VASP triple GP(5)-motif via its SH3 domain. cAMP-dependent protein kinase-mediated VASP phosphorylation at Ser157 inhibits alphaII-spectrin-VASP binding. VASP is dephosphorylated upon formation of cell-cell contacts and in confluent, but not in sparse cells, alphaII-spectrin colocalizes with nonphosphorylated VASP at cell-cell junctions. Ectopic expression of the alphaII-spectrin SH3 domain at cell-cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell-cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas alphaII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that alphaII-spectrin-VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.

SUBMITTER: Benz PM

PROVIDER: S-EPMC2213610 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes.

Benz Peter M PM Blume Constanze C Moebius Jan J Oschatz Chris C Schuh Kai K Sickmann Albert A Walter Ulrich U Feller Stephan M SM Renné Thomas T

The Journal of cell biology 20080101 1

Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified alphaII-spectrin as such a VASP-interacting protein. alphaII-Spectrin binds to the VASP triple GP(5)-motif via its SH3 domain. cAMP-dependent protein kinase-mediated VASP ...[more]

PMID: 18195108

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Project description:Calpain-catalyzed proteolysis of II-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves II spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), betaII spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second II spectrin cleavage at G1230*S1231. BetaII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the betaII spectrin COOH-terminus. CaM promotes II spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant II spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds alphaII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both alphaII and betaII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates alphaII spectrin cleavage only at Y1176*G1177, while II spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of alphaII and then betaII spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both alphaII and betaII spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of alphaII spectrin vs betaII spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses: one that enhances skeletal plasticity without destroying the spectrin-actin skeleton, characterized by preservation of betaII spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of betaII spectrin and complete dissolution of the spectrin skeleton.

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Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes.

Publications

Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes.

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