Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.)

We have isolated a full-length cDNA clone (CpCDPK1) encoding a calcium-depe ndent protein kinase (CDPK) gene from zucchini (Cucurbita pepo L.). The pre dicted amino acid sequence of the cDNA shows a remarkably high degree of si milarity to members of the CDPK gene family from Arabidopsis thaliana, espe cially AtCPK1 and AtCPK2. Northern analysis of steady-state mRNA levels for CpCPKI in etiolated and l ight-grown zucchini seedlings shows that the transcript is most abundant in etiolated hypocotyls and overall expression is suppressed by light. As described for other members of the CDPK gene family from different speci es, the CyCPK1 clone has a putative N-terminal myristoylation sequence. In this study, site-directed mutagenesis and an in vitro coupled transcription /translation system were used to demonstrate that the protein encoded by th is cDNA is specifically myristoylated by a plant N-myristoyl transferase. T his is the first demonstration of myristoylation of-a CDPK protein which ma y contribute to the mechanism by which this protein is localized to the pla sma membrane.