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Plasma membrane Ca2+-ATPase isoform 4b is phosphorylated on tyrosine 1176 in activated human platelets
Tina C Wan1, Martin Zabe, William L Dean
1Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292, USA.
Abstract:
Plasma membrane Ca(2+) -ATPase isoform 4b (PMCA4b) is phosphorylated on a tyrosine residue during platelet activation resulting in inhibition of its ATPase activity. We now report that tyrosine 1176 (Y(1176)) in the carboxyl (C-) terminal domain of PMCA4b is the phosphorylated residue. Two tyrosine residues located in the C-terminus of PMCA4b, Y(1122) and Y(1176) can be removed by calpain-dependent cleavage. This truncation removes all of the tyrosine phosphates added to PMCA during platelet activation. Sequence analysis indicates that Y(1176) is a likely substrate for focal adhesion kinase (FAK), while Y(1122) is not located in a tyrosine phosphorylation motif. This is the same residue we reported earlier to be phosphorylated by Src kinase in vitro. Thus we conclude that Y(1176) is the only tyrosine phosphorylated during platelet activation. Results of co-immunoprecipitation, treatment with tyrosine kinase inhibitors and integrin inhibition experiments suggest that FAK is responsible for PMCA4b tyrosine phosphorylation during platelet activation.
Insights
Platelet activation phosphorylates plasma membrane Ca(2+)-ATPase isoform 4b (PMCA4b) at tyrosine 1176, inhibiting its activity. Focal adhesion kinase (FAK) is identified as the primary kinase responsible for this crucial regulatory event.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Plasma membrane Ca(2+)-ATPase isoform 4b (PMCA4b) regulates intracellular calcium levels.
- PMCA4b activity is modulated by post-translational modifications, including phosphorylation.
- Tyrosine phosphorylation of PMCA4b during platelet activation leads to inhibited ATPase activity.
Purpose of the Study:
- To identify the specific tyrosine residue phosphorylated on PMCA4b during platelet activation.
- To elucidate the kinase responsible for PMCA4b tyrosine phosphorylation.
- To investigate the role of calpain cleavage in PMCA4b regulation.
Main Methods:
- Site-directed mutagenesis to mutate tyrosine residues in PMCA4b.
- Analysis of PMCA4b phosphorylation status using phospho-specific antibodies.
- Co-immunoprecipitation assays to identify interacting kinases.
- Treatment with tyrosine kinase inhibitors and integrin inhibition.
Main Results:
- Tyrosine 1176 (Y(1176)) in the C-terminal domain of PMCA4b is the sole site of tyrosine phosphorylation during platelet activation.
- Calpain-dependent cleavage at Y(1122) and Y(1176) removes all tyrosine phosphates from PMCA4b.
- Focal adhesion kinase (FAK) is identified as the primary kinase responsible for Y(1176) phosphorylation.
- Src kinase can phosphorylate Y(1176) in vitro, but FAK is implicated in vivo.
Conclusions:
- Y(1176) is the critical site for PMCA4b tyrosine phosphorylation during platelet activation, leading to functional inhibition.
- FAK-mediated phosphorylation of PMCA4b at Y(1176) is a key regulatory mechanism in platelet signaling.
- Calpain cleavage provides a mechanism to reverse PMCA4b tyrosine phosphorylation and restore activity.