STAGE-SPECIFIC ACTIVITY OF THE LEISHMANIA-MAJOR CRK3 KINASE AND FUNCTIONAL RESCUE OF A SCHIZOSACCHAROMYCES-POMBE CDC2 MUTANT

Cell cycle control by cdc2-related kinases (CRKs) is essential to the regulation of cell proliferation and developmental processes in many o rganisms, Alternating phases of growth, arrest, and differentiation ar e characteristics of the infectious cycle of many trypanosomatid paras ites, raising the possibility that members of the trypanosomatid CRK g ene family participate in the regulation of these essential processes. Here we describe properties of the CRK3 gene from Leishmania major, w hich encodes a 36 kDa protein kinase showing 60% amino acid sequence i dentity with human CDK2, including several conserved sites implicated in regulation of kinase activity. CRK3 mRNA was constitutively express ed throughout the parasite life cycle, but histone H1 kinase activity of an epitope tagged CRK3 protein was greater in log-phase than in sta tionary-phase promastigotes. When integrated into the genome and expre ssed at the optimal level, CRK3 was able to rescue the growth defect o f a Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)), indicating th at CRK3 is a functional homolog of cdc2 Mutants of CRK3 at several key regulatory residues showed the expected dominant negative effects on the S. pombe mutant. This is the first example of functional expressio n of a trypanosomatid CRK in yeast, opening the way for further geneti c studies within this amenable organism. (C) 1998 Elsevier Science Ire land Ltd. All rights reserved.