Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence.

The human pim-1 proto-oncogene was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein and the enzymatic properties of its kinase activity were characterized. Likewise, a Pim-1 mutant lacking intrinsic kinase activity was constructed by site-directed mutagenesis (Lys67 to Met) and expressed in E. coli. In vitro assays with the mutant Pim-1 kinase showed no contaminating kinase activity. The wild-type Pim-1 kinase-GST fusion protein showed a pH optimum of 7 to 7.5 and optimal activity was observed at either 10 mM MgCl2 or 5 mM MnCl2. Higher cation concentrations were inhibitory, as was the addition of NaCl to the assays. Previous work by this laboratory assaying several proteins and peptides showed histone H1 and the peptide Kemptide to be efficiently phosphorylated by recombinant Pim-1 kinase. Here we examine the substrate sequence specificity of Pim-1 kinase in detail. Comparison of different synthetic peptide substrates showed Pim-1 to have a strong substrate preference for the peptide Lys-Arg-Arg-Ala-Ser*-Gly-Pro with an almost sixfold higher specificity constant kcat/Km over that of the substrate Kemptide (Leu-Arg-Arg-Ala-Ser*-Leu-Gly). The presence of basic amino acid residues on the amino terminal side of the target Ser/Thr was shown to be essential for peptide substrate recognition. Furthermore, phosphopeptide analysis of calf thymus histone H1 phosphorylated in vitro by Pim-1 kinase resulted in fragments containing sequences similar to that of the preferred synthetic substrate peptide shown above. Therefore, under optimized in vitro conditions, the substrate recognition sequence for Pim-1 kinase is (Arg/Lys)3-X-Ser/Thr*-X', where X' is likely neither a basic nor a large hydrophobic residue.

[1]  R. Reeves,et al.  Recombinant human pim-1 protein exhibits serine/threonine kinase activity. , 1991, The Journal of biological chemistry.

[2]  A. Berns,et al.  Predisposition to lymphomagenesis in pim-1 transgenic mice: Cooperation with c-myc and N-myc in murine leukemia virus-induced tumors , 1989, Cell.

[3]  T. Hunter.  Cooperation between oncogenes , 1991, Cell.

[4]  E. Krebs,et al.  Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. , 1977, The Journal of biological chemistry.

[5]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Hagemeijer,et al.  The human pim-1 gene is not directly activated by the translocation (6;9) in acute nonlymphocytic leukemia. , 1989, Oncogene.

[7]  Susan S. Taylor,et al.  cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. , 1990, Annual review of biochemistry.

[8]  M. Lohuizen,et al.  Very high frequency of lymphoma induction by a chemical carcinogen in pim-1 transgenic mice , 1989, Nature.

[9]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[10]  A. Berns,et al.  The pim‐1 oncogene encodes two related protein‐serine/threonine kinases by alternative initiation at AUG and CUG. , 1991, The EMBO journal.

[11]  T. Hunter,et al.  The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. , 1988, Science.

[12]  Jos Domen,et al.  The primary structure of the putative oncogene pim-1 shows extensive homology with protein kinases , 1986, Cell.

[13]  L. Nagarajan,et al.  The human PIM-1 gene product is a protein serine kinase. , 1991, Cancer research.

[14]  L. Liao,et al.  The amino acid sequence of residues 1-104 of CTL-1, a bovine H1 histone. , 1981, The Journal of biological chemistry.

[15]  R. Reeves,et al.  Microheterogeneity of the mammalian high-mobility group proteins 14 and 17 investigated by reverse-phase high-performance liquid chromatography. , 1985, Analytical biochemistry.

[16]  D. Glass.  [13] Synthesis of oligopeptides for the study of cyclic nucleotide-dependent protein kinases , 1983 .

[17]  R. Roskoski.  [1] Assays of protein kinase , 1983 .

[18]  A. Mildvan,et al.  Mechanistic studies of cAMP-dependent protein kinase action. , 1984, CRC critical reviews in biochemistry.

[19]  C. Morris,et al.  Basophils (Bsp-1+) derive from the leukemic clone in human myeloid leukemias involving the chromosome breakpoint 9q34. , 1989, Blood.

[20]  D. Wells,et al.  Histone and histone gene compilation and alignment update. , 1991, Nucleic acids research.

[21]  A. Berns,et al.  Comparison of the human and mouse PIM-1 cDNAs: nucleotide sequence and immunological identification of the in vitro synthesized PIM-1 protein. , 1987, Oncogene research.

[22]  S. C. Rall,et al.  Amino acid sequence and sequence variability of the amino-terminal regions of lysine-rich histones. , 1971, The Journal of biological chemistry.

[23]  A. Berns,et al.  Mice bearing the E mu-myc and E mu-pim-1 transgenes develop pre-B-cell leukemia prenatally , 1991, Molecular and cellular biology.

[24]  D. Givol,et al.  The cDNA sequence and gene analysis of the human pim oncogene. , 1987, Gene.

[25]  D. Wells,et al.  A comprehensive compilation and alignment of histones and histone genes. , 1989, Nucleic acids research.

[26]  T. Langan.  Action of adenosine 3',5'-monophosphate-dependent histone kinase in vivo. , 1969, The Journal of biological chemistry.

[27]  M. Power,et al.  Primary structure of the putative human oncogene, pim-1. , 1990, Gene.

[28]  J. Zheng,et al.  Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[29]  D. Smith,et al.  Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. , 1988, Gene.

[30]  C. Gibbs,et al.  Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase. , 1991, Biochemistry.

[31]  T. Meeker,et al.  Cloning and characterization of the human PIM‐1 gene: A putative oncogene related to the protein kinases , 1987, Journal of cellular biochemistry.

[32]  A Ma,et al.  E mu N- and E mu L-myc cooperate with E mu pim-1 to generate lymphoid tumors at high frequency in double-transgenic mice. , 1991, Oncogene.

[33]  D. Givol,et al.  Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity , 1988, Molecular and cellular biology.

[34]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.