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Polycation-dependent, Ca2+-antagonized phosphorylation of calmodulin by casein kinase-2 and a spleen tyrosine protein
Abstract:
Ten distinct protein kinases have been tested for their ability to phosphorylate calmodulin. Only casein kinase-2 and a spleen tyrosine protein kinase (TPK-III) proved effective, their phosphorylation efficiency being dramatically enhanced by histones and other polybasic peptides while being depressed by 50 microM Ca2+. Phosphorylation by CK-2 takes place with a Km of 12 microM calmodulin, leading to the incorporation of more than 1.5 mol P/mol substrate. Ser81 and Thr79 are among the residues affected. On the other hand, the two tyrosyl residues of calmodulin are both phosphorylated by TPK-III, Tyr99 being preferred over Tyr138.
Insights
Two protein kinases, casein kinase-2 and spleen tyrosine protein kinase (TPK-III), effectively phosphorylate calmodulin. Their activity is modulated by polybasic peptides and calcium ions, affecting specific residues.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Calmodulin is a crucial calcium-binding protein involved in numerous cellular signaling pathways.
- Protein phosphorylation is a key post-translational modification regulating protein function.
- Understanding calmodulin phosphorylation is vital for deciphering cellular regulation.
Purpose of the Study:
- To identify protein kinases capable of phosphorylating calmodulin.
- To investigate the regulatory factors influencing calmodulin phosphorylation.
- To characterize the specific sites of calmodulin phosphorylation by identified kinases.
Main Methods:
- Screening of ten distinct protein kinases for calmodulin phosphorylation activity.
- Assessing the impact of histones, polybasic peptides, and calcium ions on phosphorylation efficiency.
- Kinetic analysis (Km determination) of casein kinase-2 phosphorylation.
- Identification of phosphorylated residues using biochemical techniques.
Main Results:
- Casein kinase-2 (CK-2) and spleen tyrosine protein kinase (TPK-III) were identified as effective calmodulin-phosphorylating enzymes.
- Phosphorylation efficiency by both kinases was significantly enhanced by polybasic peptides and inhibited by calcium ions (50 microM).
- CK-2 phosphorylated calmodulin with a Km of 12 microM, affecting Ser81 and Thr79 residues.
- TPK-III phosphorylated both tyrosyl residues (Tyr99 and Tyr138) of calmodulin, with a preference for Tyr99.
Conclusions:
- CK-2 and TPK-III are novel calmodulin-modifying enzymes.
- Polybasic peptides and calcium ions play critical roles in regulating calmodulin phosphorylation.
- Specific phosphorylation sites identified provide insights into calmodulin's functional regulation by these kinases.