IDENTIFICATION OF PLECTIN AS A SUBSTRATE OF P34(CDC2) KINASE AND MAPPING OF A SINGLE PHOSPHORYLATION SITE

Plectin is an in vitro substrate for various kinases present in cell l ysates from mitotic and interphase Chinese hamster ovary cells. Sensit ivity of plectin kinase activity to the inhibitor olomoucine, and two- dimensional tryptic peptide mapping of plectin phosphorylated by vario us kinase preparations suggested that the major plectin kinase activit y in mitotic extracts is related to the cell cycle regulator kinase p3 4(cdc2). Bacterial expression of various truncated plectin mutant prot eins comprising different domains of the molecule and their phosphoryl ation by purified p34(cdc2) kinase revealed that the target site of th is kinase resided within plectin's C-terminal globular domain. Among t he subdomains of the C-terminal region (six repeats and a short tail s equence), only repeat 6 and the tail were phosphorylated by p34(cdc2) kinase. As shown by two-dimensional phosphopeptide mapping, repeat 6, but not the tail, contained a mitosis-specific phosphorylation site ta rgeted by p34(cdc2) kinase in intact plectin molecules. By performing site-directed mutagenesis of a potential p34(cdc2) recognition sequenc e motif within the repeat 6 domain, threonine 4542 was identified as t he major target for the kinase. Protein kinase A, phosphorylating plec tin also within repeat 6, targeted sites that were clearly different f rom those of p34(cdc2) kinase.