Tyrosine Phosphorylation of α-Actinin in Activated Platelets

Abstract

The integrin ␣ IIb ␤ 3 mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human ␣-actinin sequences deposited in the Swiss Protein Database. ␣-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with ␣-actinin and anti-phosphotyrosine antibodies. The distribution of ␣-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained ␣-actinin. The purified ␣-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. ␣-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with ␣-actinin. Taken together, these data demonstrate that a fraction of ␣-actinin is tyrosine-phosphorylated in activated platelets.Tyrosine phosphorylation of protein substrates is central to all cellular processes that are regulated by the integrin family of extracellular matrix receptors (1) including cell shape change, migration, growth, and survival (for recent reviews see Refs. 2-6). The array of responses regulated by integrins is greatly simplified in platelets that are enucleated, terminally differentiated cells. Platelets, therefore, provide a useful model system to study integrin-mediated protein tyrosine phosphorylation events that are closely linked to cell shape change and the underlying cytoskeleton organization.A rapid increase in protein tyrosine phosphorylation is detected within seconds to minutes of platelet activation by soluble agonists such as thrombin or phorbol 12-myristate 13-acetate (PMA) 1 (7,8). Platelet adhesion to immobilized matrix proteins such as fibrinogen triggers similar tyrosine phosphorylation events (9). Included in the first group of proteins that become tyrosine-phosphorylated are the non-receptor tyrosine kinases pp72syk (10) and RFTK/PYK (11), the guanine nucleotide exchange factor Vav (12), and the mitogen-activated protein (MAP) kinase family members extracellular signal-regulated kinase 2 (ERK2) and p38 (13,14). In platelets stimulated by thrombin, PMA, or adhesion to fibrinogen, a second wave of protein tyrosine phosphorylation that is strictly dependent on both ligand binding to the ␣ IIb ␤ 3 integrin receptor and the cytoskeleton organization is observed (7). As such, tyrosine phosphorylation of this ...

Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells

“…This regulation of the myosin heavy chain differs from the usual regulation through the light chain ( Conti and Adelstein, 2008 ). Talin, ␣ -actinin, and the actin nucleator mDia1 also appeared regulated (to a lesser extent), and all have been implicated previously in phagocytosis (respectively: [( Turner et al, 1989 ;Greenberg et al, 1990 ); ( Crowley and Horwitz, 1995 ;Izaguirre et al, 1999 ); ( Meng et al, 2004 ;Brandt et al, 2007 )]). Although phospho-paxillin was downregulated in our imaging studies, it was not prominent in our immunoblots and was unaffected by blebbistatin, which otherwise mimicked CD47 ' s inhibitory effects ( Figs.…”

“…8 C , bottom). Additional cytoskeletal proteins that were detected in the macrophages and might be targeted in the CD47-SIRP ␣ phosphotyrosine pathway include Talin-1 ( Turner et al, 1989 ;Greenberg et al, 1990 ), mDIA1 ( Meng et al, 2004 ), and ␣ -actinin ( Crowley and Horwitz, 1995 ;Izaguirre et al, 1999 ). Capabilities of MS to identify phosphotyrosine are currently limited, as here, by low sequence coverage and sub-stoichiometric phosphorylation ( McLachlin and Chait, 2001 ).…”

Focal and Segmental Glomerulosclerosis in Mice with Podocyte-Specific Expression of Mutant α-Actinin-4

“…COS-7 cells were transiently transfected with pCDNA3 vector, HA-ACTN4 wt, or HA-ACTN4 mutant. The actin cytoskeleton was pelleted from cell lysates at 10 5 ϫ g (23) and the amount of HA-␣-actinin-4 assessed by Western blot as analyzed with a polyclonal anti-HA tag antibody (1:1000). As shown in Figure 2b, compared to the wt form, a significantly greater proportion of mutant murine ␣-actinin-4 associated with the insoluble cytoskeletal fraction containing an equal amount of pelleted actin.…”

The Cytoskeletal/Non-muscle Isoform of α-Actinin Is Phosphorylated on Its Actin-binding Domain by the Focal Adhesion Kinase

“…We have recently reported that ␣-actinin is tyrosine-phosphorylated in activated platelets (1). Tyrosine phosphorylation of ␣-actinin was also observed in activated T-cells (31).…”