Isolation and characterization of a cDNA coding cowpea (Vigna unguiculata (L.) Walp.) calcineurin B-like protein-interacting protein kinase, VuCIPK1

Abstract

We isolated a calcineurin B-like protein (CBL)-interacting protein kinase (EC 2.7.11.1) homolog (VuCIPK1) from cowpea (Vigna unguiculata (L.) Walp.) with significant similarity to AtCIPK3 (86% amino acid identity), which is involved in K⁺ transport. VuCIPK1 mRNA was detected in the whole plant by RT-PCR. Any significant change in VuCIPK1 mRNA levels was not detected when cowpea was subjected to various environmental stresses. Immunoblot assay with an anti-VuCIPK1 specific antibody and an anti-CBL antibody showed that immunologically CIPK- and CBL-related polypeptides were preferentially associated with membrane fractions of cowpea. The anti-VuCIPK1 antibody cross-reacted to two different CIPK-related polypeptides with relative molecular masses of 51 kDa in root and 55 kDa in mature leaf. Immunoprecipitation assay by anti-VuCIPK1 antibody using cowpea leaf extracts showed that endogenous VuCIPK1 was autophosphorylated on the threonine residues in response to salt stress. The recombinant VuCIPK1 phosphorylated casein in the presence of Mn²⁺. These observations suggest that membrane-associated VuCIPK1 is involved osmotic stress-activated kinase regulated by its phosphorylation status.