Kevin D. Lustig

Systematic identification of mitotic phosphoproteins

BACKGROUND Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. RESULTS Using a screen that scores for phosphorylation of proteins, which were translated from pools of cDNA plasmids in vitro, by either phosphoepitope antibody recognition or electrophoretic mobility shifts, we have identified 20 mitotically phosphorylated proteins from Xenopus embryos, 15 of which have sequence similarity to other proteins. Of these proteins, five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. CONCLUSIONS Examination of this broad set of mitotic phosphoproteins has allowed us to draw three conclusions about how the activation of CDKs regulates cell-cycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation. Third, the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression.

Specific Proteolysis of the Kinase Protein Kinase C-related Kinase 2 by Caspase-3 during Apoptosis IDENTIFICATION BY A NOVEL, SMALL POOL EXPRESSION CLONING STRATEGY

The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translatedin vitro in the presence of [35S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp117 and Asp700 in vitro. In addition, PRK2 is cleaved rapidly during Fas- and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.

Small pool expression screening: Identification of genes involved in cell cycle control, apoptosis, and early development

Publisher Summary This chapter discusses the identification of genes involved in cell cycle control, apoptosis, and early development. Traditional genetic and biochemical methods have been quite successful in identifying genes that are essential for cell cycle progression and early embryonic development, among other diverse biological processes. Nevertheless, only a small fraction of the genes in the vertebrate genome has been functionally characterized. This chapter describes a systematic and broadly applicable approach to cloning genes based solely on the biological activities or biochemical properties of the gene products. It describes several potential applications of this expression cloning approach, and also discusses its use in related types of screening procedures. It describes general methods used to prepare library pools of cDNA, RNA, and protein, and the sib selection techniques used to subdivide a pool once it is found to contain a candidate activity.