ATPK1, A NOVEL RIBOSOMAL-PROTEIN KINASE GENE FROM ARABIDOPSIS .2. FUNCTIONAL AND BIOCHEMICAL-ANALYSIS OF THE ENCODED PROTEIN

The Arabidopsis Atpk1 protein expressed in insect cells and plant cell s exhibited multiple sizes consisting mainly of two doublets: p70 (68 and 70 kDa) and p85 (82 and 85 kDa). Extraction of p85 from cells requ ired the presence of SDS, suggesting that p85 is associated with less soluble subcellular components. p70 was extracted by nonionic detergen t without SDS, indicating that this form is cytoplasmic. p70 expressed in either Arabidopsis or insect cells underwent serine-specific autop hosphorylation, indicating that Atpk1 is a protein-serine kinase. A po int mutation (lysine 163 to arginine) in the ATP-binding site of the c atalytic domain substantially diminished activity when expressed in in sect cells. A 14-kDa protein (p14) was co-immunoprecipitated with p70 from insect cells expressing wild-type Atpk1 and was phosphorylated in immune complex kinase assays with Atpk1, suggesting it is a homolog o f a natural substrate of Atpk1. Two plant ribosomal proteins (14 and 1 6 kDa) can be phosphorylated by the Atpk1 protein kinase, and we propo se that Atpk1 is a novel ribosomal protein kinase. A 60-kDa form of At pk1 derived from the insect cell-expressed p70 was more highly phospho rylated than p70 in in vitro kinase assays, suggesting a negative regu latory domain can be removed by proteolysis.