title: Recombinant human pim-1 protein exhibits serine/threonine kinase activity.
abstract: The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.
authors.affiliation: Program in Genetics and Cell Biology, Washington State University, Pullman 99164.
authors.forename: D
authors.initials: D
authors.lastname: Hoover
authors.forename: M
authors.initials: M
authors.lastname: Friedmann
authors.forename: R
authors.initials: R
authors.lastname: Reeves
authors.forename: N S
authors.initials: NS
authors.lastname: Magnuson
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051573:Proto-Oncogene Proteins c-pim-1; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D011494:Protein Kinases; C496634:PIM1 protein, human; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1