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 b y which caspases induce cell death have been greatly hindered by the l ack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to ra pidly isolate cDNAs encoding caspase substrates that are cleaved durin g apoptosis, Small cDNA pools (approximately 100 clones each) are tran scribed/translated in vitro in the presence of [S-35]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells, cDNA pools encoding proteins that are sp ecifically cleaved by the apoptotic extract and whose cleavage is prev ented by the caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketo ne are subdivided and retested until a single cDNA is isolated, Using this approach, we isolated a partial cDNA encoding protein kinase C-re lated kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp(117) and As p(700) in vitro, In addition, PRK2 is cleaved rapidly during Fas-and s taurosporine-induced apoptosis in vitro by caspase-3 or a closely rela ted caspase, Both of the major apoptotic cleavage sites of PRK2 in viv o lie within its regulatory domain, suggesting that its activity may b e deregulated by proteolysis.