Abstract
By using transient elevations of cytosolic free calcium levels triggered by integrin antibody or laminin (Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443), we have demonstrated that protein phosphatase 2A (PP2A) is implicated in the regulation of reversible phosphorylation of integrin. In E63 skeletal myoblasts, the treatment of PP2A inhibitors such as okadaic acid and endothall induces an increase of phosphorylation of integrin beta1A and thereby inhibits integrin-induced elevation of cytosolic calcium level and formation of focal adhesions. None of these effects were in differentiated myotubes expressing the alternate beta1D isoform. In the presence of okadaic acid, PP2A in association with integrin beta1A was reduced on myoblasts, whereas beta1D on myotubes remained bound with PP2A. Both co-immunoprecipitation and in vitro phosphatase assays revealed that dephosphorylation of residues Thr788-Thr789 in the integrin beta1A cytoplasmic domain is dependent upon PP2A activity. Mutational analysis of the cytoplasmic domain and confocal microscopy experiments indicated that substitution of Thr788-Thr789 with Asn788-Asn789 is of critical importance for regulating the function of integrin beta1. These results suggest that PP2A may be a primary regulator of threonine phosphorylation of integrin beta1A and subsequent activation of downstream signaling molecules. Taken together, we propose that dephosphorylation of residues Thr788-Thr789 in the cytoplasmic domain of integrin beta1A may contribute to the linkage of integrins to focal adhesion sites and induce the association with cytoskeleton proteins. The switch of integrin beta1A to beta1D isoform in myotubes therefore may be a mechanism to escape from phospho-regulation by PP2A and promotes a more stable association of the cytoskeleton with the extracellular matrix.
Highlights
Integrins are heterodimeric transmembrane proteins consisting of ␣ and  subunits and mediate many of the interactions between adjacent cells or between cells and extracellular matrix (ECM).1 The specificity of ligand binding by integrins is
The 4-day-old cultured cell treated with 100 nM Okadaic acid (OA) and 100 M ETL were immunoprecipitated with anti-integrin 1A antibody and followed by immunoblotting with anti-phosphothreonine (P-thr788/789) antibody
Our studies demonstrate that pretreatment of protein serine/threonine phosphatase inhibitors such as OA or ETL inhibits the elevation of [Ca2ϩ]i and formation of focal adhesions
Summary
Integrins are heterodimeric transmembrane proteins consisting of ␣ and  subunits and mediate many of the interactions between adjacent cells or between cells and extracellular matrix (ECM). The specificity of ligand binding by integrins is. In recent studies [10, 11], more cellular proteins have been reported to directly or indirectly interact with the cytoplasmic domain of integrin 1 Cytoskeletal proteins such as ␣-actinin, F-actin, skelemin, talin, vinculin, filamin, and tensin are known to bind the integrin 1 cytoplasmic domain [12,13,14,15]. Signaling molecules such as focal adhesion kinase, integrin-linked kinase, phosphoinositide 3-kinase, Src, Cas, and novel proteins such as TAP 20 are targeted to adhesion sites where they come into contact with the integrin cytoplasmic domain (16 –24). The Thr788–Thr789 residues, potential phosphorylation sites in the integrin 1A cytoplasmic domain, are substituted with Asn788–Asn789 in the 1D splice variant
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