CHARACTERIZATION OF AN ARABIDOPSIS CALMODULIN-LIKE DOMAIN PROTEIN-KINASE PURIFIED FROM ESCHERICHIA-COLI USING AN AFFINITY SANDWICH TECHNIQUE

A full-length cDNA encoding a calcium-dependent protein kinase with a calmodulin-like domain from Arabidopsis thaliana (AK-1 for Arabidopsis kinase-1) has been expressed as a fusion protein (called AK-1-6H) in Escherichia coil and purified to near homogeneity with high specific a ctivity (typically 2000 nmor min(-1) mg(-1)) using an ''affinity sandw ich'' technique. AK-1-6H protein phosphorylation activity using histon e as substrate was stimulated up to 50-fold by the addition of calcium alone or up to 5-fold by the addition of specific phospholipids alone ; together calcium and these lipids acted synergistically to give up t o 100-fold stimulation. We earlier reported that, of a wide array of l ipids tested, only phosphatidylinositol and lysophosphatidylcholine st imulated histone phosphorylation by AK-1 [Harper, J. F., Binder, B. M. , and Sussman M. R. (1993) Biochemistry 32, 3282-3290]. The properties of lipid stimulation were further explored by testing the effects of lipids on autophosphorylation and on other catalytic properties of the kinase. Although phosphatidylinositol stimulated autophosphorylation up to 11-fold, lysophosphatidylcholine was inactive. Basic peptides su ch as polylysine (average M(r) similar to 37 100) were potent, mixed-t ype inhibitors of AK-1-6H with an IC50 of 2 nM. In the presence of pho sphatidylinositol, the inhibition was reduced and the IC50 for polylys ine was increased to 341 nM. As with autophosphorylation, lysophosphat idylcholine was inactive in alleviating the basic peptide inhibition, which suggests that this lipid's stimulatory effects using exogenous s ubstrate are distinct from those of phosphatidylinositol. These result s are consistent with a model in which phosphoinositides directly inte ract with the kinase protein and alleviate a catalytic block caused by basic charges.