Genetic evidence from retinoblastoma patients and experiments describing the mechanism of cellular transformation by the DNA tumor viruses have defined a central role for the retinoblastoma protein (pRB) family of tumor suppressors in the normal regulation of the eukaryotic cell cycle. These proteins, pRB, p107, and p130, act in a cell cycle-dependent manner to regulate the activity of a number of important cellular transcription factors, such as the E2F-family, which in turn regulate expression of genes whose products are important for cell cycle progression. In addition, inhibition of E2F activity by the pRB family proteins is required for cell cycle exit after terminal differentiation or nutrient depletion. The loss of functional pRB, due to mutation of both RB1 alleles, results in deregulated E2F activity and a predisposition to specific malignancies. Similarly, inactivation of the pRB family by the transforming proteins of the DNA tumor viruses overcomes cellular quiescence and prevents terminal differentiation by blocking the interaction of pRB, p107, and p130 with the E2F proteins, leading to cell cycle progression and, ultimately, cellular transformation. Together these two lines of evidence implicate the pRB family of negative cell cycle regulators and the E2F family of transcription factors as central components in the cell cycle machinery.

Pim-1 is an oncogene-encoded serine-threonine kinase that is expressed primarily in cells of the hematopoietic system and germ line. The full-length coding regions of both human and Xenopus laevis Pim-1 were expressed as recombinant bacterial fusion proteins that autophosphorylated in vitro and exhibited phosphotransferase activity towards various exogenous substrates. The consensus sequence for phosphorylation by Pim-1 was defined by stepwise replacement of the amino acids in peptide substrate analogues based on the carboxyl-terminal segment of human ribosomal protein S6 (residues 229-249). The optimal substrate peptide for Pim-1 was determined to be Lys/Arg-Lys/Arg-Arg-Lys/Arg-Leu-Ser/Thr-X, where X is an amino acid residue with a small side chain. These results were confirmed using X. laevis Pim-1 expressed in COS cells. These findings could permit the identification of physiological substrates of Pim-1 and predict the location of phosphorylation sites within these proteins.

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.

Mitogen activated protein (MAP) kinases and their target ribosomal protein S6 (RSK) kinases have been recognized as shared components in the intracellular signaling pathways of many diverse cytokines. Recent studies have extended this protein kinase cascade by identifying the major activator of vertebrate MAP kinases as a serine/threonine/tyrosine-protein kinase called MEK, which is related to yeast mating factor-regulated protein kinases encoded by the STE7 and byr1 genes. MEK, in turn, may be activated following its phosphorylation on serine by either of the kinases encoded by proto-oncogenes raf1 or mos, as well as by p78mekk, which is related to the yeast STE11 and byr2 gene products. Isoforms of all of these protein kinases may specifically combine to assemble distinct modules for intracellular signal transmission. However, the fundamental architecture of these protein kinase cascades has been highly conserved during eukaryotic evolution.

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Pim-1 is an oncogene-encoded serine-threonine kinase that is expressed primarily in cells of the hematopoietic system and germ line. The full-length coding regions of both human and Xenopus laevis Pim-1 were expressed as recombinant bacterial fusion proteins that autophosphorylated in vitro and exhibited phosphotransferase activity towards various exogenous substrates. The consensus sequence for phosphorylation by Pim-1 was defined by stepwise replacement of the amino acids in peptide substrate analogues based on the carboxyl-terminal segment of human ribosomal protein S6 (residues 229-249). The optimal substrate peptide for Pim-1 was determined to be Lys/Arg-Lys/Arg-Arg-Lys/Arg-Leu-Ser/Thr-X, where X is an amino acid residue with a small side chain. These results were confirmed using X. laevis Pim-1 expressed in COS cells. These findings could permit the identification of physiological substrates of Pim-1 and predict the location of phosphorylation sites within these proteins.

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