The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth.

The 78-kDa protein kinase Mekk1 plays an important role in the stress response pathway that involves the activation of downstream kinases Sek1 and stress-activated protein kinase/c-Jun NH 2 -terminal kinase. Conserved serine and threonine residues located between the kinase subdomains VII and VIII of many protein kinases are phosphorylated for maximal kinase activation. Two threonine residues within this region in Mekk1 at positions 560 and 572, but not the serine at 557, were shown to be essential for catalytic activity in this study. When these threonine residues were replaced with alanine, there was a significant loss in phosphotransferase activity toward the primary substrate, Sek1, and a large decrease in autophosphorylation activity. Site-directed mutagenesis demonstrated that these threonine residues cannot be replaced with either serine or glutamic acid for preservation of phosphotransferase activity. Further examination of the Mekk1 mutants isolated from 32 P-labeled transfected COS cells showed that Thr-560 and Thr-572 were indeed phosphorylated after two-dimensional tryptic-chymotryptic phosphopeptide analysis. Additional determinants in the NH 2 -terminal domain of Mekk1 also play a role in the regulation of Mekk1 activity. Although Pak3 and PKC can activate Mekk1 in vivo, this interaction is indirect and independent, since there was no direct phosphorylation of Mekk1 by Pak3 or PKC or of Pak3 by PKC, respectively.

Pim-1 is an oncogene-encoded serine/threonine kinase expressed primarily in cells of the hematopoietic and germ line lineages. Previously identified only in mammals, pim-1 cDNA was cloned and sequenced from the African clawed frog Xenopus laevis. The coding region of Xenopus pim-1 encoded a protein of 324 residues, which exhibited 64% amino acid identity with the full-length human cognate. Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and in COS cells. Phosphoamino acid analysis revealed that recombinant Pim-1 autophosphorylated on serine and threonine and to a more limited extent on tyrosine. Electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites, and the primary autophosphorylation site of GST-Pim-1 was identified as Ser-190 with Thr-205 and Ser-4 being minor sites. Ser-190, which immediately follows the high conserved Asp-Phe-Gly motif in catalytic subdomain VII, is also featured in more than 20 other protein kinases. To evaluate the importance of the Ser-190 site on the phosphotransferase activity of Pim-1, Ser-190 was mutated to either alanine or glutamic acid, and the constructs were expressed in bacteria as GST fusion proteins and in COS cells. These mutants confirmed that Ser-190 is a major autophosphorylation site of Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 residue may serve to activate this kinase.

We have examined the human Cyr61 gene and its expression in normal fibroblasts. The core promoter, second intron, and 3' untranslated region (UTR) are highly conserved between the human and mouse genes. Cyr61 expression was induced slightly slower but more transiently in human fibroblasts compared to Rat-2 fibroblasts. These differences may relate to the absence of a serum response element in the human Cyr61 promoter, and the presence of additional AU-rich elements within the 3' UTR. Cycloheximide causes accumulation of human Cyr61 RNA in the absence of growth factors, and EGF prevents decay of transcripts in actinomycin-D-treated cells, which suggests that induction by growth factors may partially involve mRNA stabilization. We detect an alternative RNA in serum-stimulated fibroblasts containing an in-frame deletion within exon 4 which disrupts the thrombospondin type 1 repeat. Constitutive expression of the full hCyr61 genomic DNA in rodent fibroblasts causes production of multiple protein species, whereas expression of hCyrDelta4 produces a single stable protein of the expected size. We also observed multiple hCyr61 protein species in normal fibroblasts following serum stimulation, indicating that Cyr61 may normally be produced as alternative isoforms.