No abstract

p21-activated protein kinase ␥-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant ␥-PAK into two peptides; 1-212 contains the majority of the regulatory domain whereas 213-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with [␥-32 P]ATP. Peptide 1-212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after autophosphorylation on threonine. Following caspase cleavage, a significant lag (ϳ5 min) is observed before autophosphorylation and activity are detected. When ␥-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After autophosphorylation of ␥-PAK in the presence of Cdc42(GTP␥S) or histone 4, both cleavage products contain phosphate and ␥-PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of ␥-PAK via cleavage by CPP32 is a two-step mechanism wherein autophosphorylation of the regulatory domain is a priming step, and activation coincides with autophosphorylation of the catalytic domain.

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous ␥-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant ␥-PAK, while native ␥-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of ␥-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant ␥-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant ␥-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, ␥-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated ␥-PAK, six of which are in common with those previously reported in ␣-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the ␥-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with ␥-PAK activation.

We have examined the properties of two Drosophila RNA polymerase II mutants, C4 and S1, during elongation, pyrophosphorolysis, and DmS-II-stimulated transcript cleavage. The C4 and S1 mutants contain a single amino acid substitution in the largest and second largest subunits, respectively. Compared with wild type, C4 had a lower elongation rate and was less efficient at reading through intrinsic elongation blocks. S1 had a higher elongation rate than wild type and was more efficient at reading through the same blocks. During elongation, C4 and wild type responded similarly to DmS-II and NH4+ whereas the S1 mutant was less responsive to both. Differences between the two mutants also appeared during DmS-II-mediated transcript cleavage and pyrophosphorolysis. During extended pyrophosphorolysis, S1 polymerase was fastest and C4 polymerase was slowest at generating the final pattern of shortened transcripts. S1 and wild type were equal in the rate of extended DmS-II mediated transcript cleavage, and C4 was slower. Our results suggest that the S1 mutation increases the time spent by the polymerase in elongation competent mode and that the C4 mutation may affect the movement of the polymerase.

Pak2 is a serine/threonine kinase that participates in the cellular response to stress. Among the potential substrates for Pak2 is the protein Myc, encoded by the proto-oncogene MYC. Here we demonstrate that Pak2 phosphorylates Myc at three sites (T358, S373, and T400) and affects Myc functions both in vitro and in vivo. Phosphorylation at all three residues reduces the binding of Myc to DNA, either by blocking the requisite dimerization with Max (through phosphorylation at S373 and T400) or by interfering directly with binding to DNA (through phosphorylation at T358). Phosphorylation by Pak2 inhibits the ability of Myc to activate transcription, to sustain cellular proliferation, to transform NIH 3T3 cells in culture, and to elicit apoptosis on serum withdrawal. These results indicate that Pak2 is a negative regulator of Myc, suggest that inhibition of Myc plays a role in the cellular response to stress, and raise the possibility that Pak2 may be the product of a tumor suppressor gene.