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Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea

Prakash, Syam SR and Jayabaskaran, Chelliah (2006) Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea. In: Journal of Plant Physiology, 163 (11). pp. 1083-1093.

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Abstract

In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as $Ca^{2+}$ -dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of $Ca^{2+}$. The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the \gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including $Ca^{2+}$ sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8–8.6 and $35–42 ^oC$, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2–9 and $ 35–42^oC$, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in $Ca^{2+}$ -mediated signaling in chickpea plants.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Elsevier.
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 13 May 2008
Last Modified: 19 Sep 2010 04:44
URI: http://eprints.iisc.ernet.in/id/eprint/13953

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