Structure and Substrate Specificity of the Pim-1 Kinase*

The Pim kinases are a family of three vertebrate protein serine/threonine kinases (Pim-1, -2, and -3) belonging to the CAMK (calmodulin-dependent protein kinase-related) group. Pim kinases are emerging as important mediators of cytokine signaling pathways in hematopoietic cells, and they contribute to the progression of certain leukemias and solid tumors. A number of cytoplasmic and nuclear proteins are phosphorylated by Pim kinases and may act as their effectors in normal physiology and in disease. Recent crystallographic studies of Pim-1 have identified unique structural features but have not provided insight into how the kinase recognizes its target substrates. Here, we have conducted peptide library screens to exhaustively determine the sequence specificity of active site-mediated phosphorylation by Pim-1 and Pim-3. We have identified the major site of Pim-1 autophosphorylation and find surprisingly that it maps to a novel site that diverges from its consensus phosphorylation motif. We have solved the crystal structure of Pim-1 bound to a high affinity peptide substrate in complexes with either the ATP analog AMP-PNP or the bisindolylmaleimide kinase inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide HCl. These structures reveal an unanticipated mode of recognition for basic residues upstream of the phosphorylation site, distinct from that of other kinases with similar substrate specificity. The structures provide a rationale for the unusually high affinity of Pim kinases for peptide substrates and suggest a general mode for substrate binding to members of the CAMK group.

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

[2]  P. Koskinen,et al.  Pim‐1 kinase promotes inactivation of the pro‐apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site , 2004, FEBS letters.

[3]  R. Eisenman,et al.  Pim-1 kinase and p100 cooperate to enhance c-Myb activity. , 1998, Molecular cell.

[4]  F. Giles.  A Pim kinase inhibitor, please , 2005 .

[5]  S. Dhanasekaran,et al.  Delineation of prognostic biomarkers in prostate cancer , 2001, Nature.

[6]  P. Koskinen,et al.  Cutting Edge: Transcriptional Activity of NFATc1 Is Enhanced by the Pim-1 Kinase1 , 2002, The Journal of Immunology.

[7]  F. Sigaux,et al.  The human protooncogene product p33pim is expressed during fetal hematopoiesis and in diverse leukemias. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Ruben Abagyan,et al.  ICM—A new method for protein modeling and design: Applications to docking and structure prediction from the distorted native conformation , 1994, J. Comput. Chem..

[9]  D S Lawrence,et al.  Definition of Optimal Substrate Recognition Motifs of Ca2+-Calmodulin-dependent Protein Kinases IV and II Reveals Shared and Distinctive Features* , 1998, The Journal of Biological Chemistry.

[10]  A. Berns,et al.  Pim-2 transgene induces lymphoid tumors, exhibiting potent synergy with c-myc , 1997, Oncogene.

[11]  T. Hunter,et al.  The Protein Kinase Complement of the Human Genome , 2002, Science.

[12]  Susan S. Taylor,et al.  cAMP‐dependent protein kinase: Crystallographic insights into substrate recognition and phosphotransfer , 1994, Protein science : a publication of the Protein Society.

[13]  Yoshihisa Suzuki,et al.  Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma. , 2005, Journal of molecular biology.

[14]  P. Cohen,et al.  Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase‐1 and p70 S6 kinase , 1996, FEBS letters.

[15]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[16]  Anton Berns,et al.  High-throughput retroviral tagging to identify components of specific signaling pathways in cancer , 2002, Nature Genetics.

[17]  M. Nissen,et al.  Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence. , 1992, Archives of biochemistry and biophysics.

[18]  L. Johnson,et al.  The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases , 1999, Nature Cell Biology.

[19]  H. Ohno,et al.  Molecular anatomy of BCL6 translocations revealed by long-distance polymerase chain reaction-based assays. , 2000, Cancer research.

[20]  M. Nawijn,et al.  Pim serine/threonine kinases regulate the stability of Socs-1 protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  L. Johnson,et al.  The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition , 1997, The EMBO journal.

[22]  T. Möröy,et al.  The serine/threonine kinase Pim-1. , 2005, The international journal of biochemistry & cell biology.

[23]  D. Graves,et al.  Studies on the specificity of phosphorylase kinase using peptide substrates. , 1977, The Journal of biological chemistry.

[24]  P. Hammerman,et al.  The Pim kinases control rapamycin-resistant T cell survival and activation , 2005, The Journal of experimental medicine.

[25]  P. Cohen,et al.  A non-radioactive method for the assay of many serine/threonine-specific protein kinases. , 2002, The Biochemical journal.

[26]  Michael B Yaffe,et al.  MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. , 2005, Molecular cell.

[27]  Wenyi Wei,et al.  Phosphorylation of the cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase. , 2002, Biochimica et biophysica acta.

[28]  Toshiyuki Obata,et al.  Peptide and Protein Library Screening Defines Optimal Substrate Motifs for AKT/PKB* , 2000, The Journal of Biological Chemistry.

[29]  S. Ness,et al.  Pim-1 Phosphorylates the DNA Binding Domain of c-Myb , 2003, Cell cycle.

[30]  H. Bessler,et al.  Increased Expression of the hPim-2 Gene In Human Chronic lymphocytic Leukemia and Non-Hodgkin Lymphoma , 2004, Leukemia & lymphoma.

[31]  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.

[32]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Likelihood-enhanced Fast Rotation Functions Biological Crystallography Likelihood-enhanced Fast Rotation Functions , 2003 .

[33]  M. Gold,et al.  CD40 Signaling in B Cells Regulates the Expression of the Pim-1 Kinase Via the NF-κB Pathway1 , 2002, The Journal of Immunology.

[34]  L. Chodosh,et al.  The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor. , 2003, Genes & development.

[35]  J. Herrero,et al.  Kinase peptide specificity: improved determination and relevance to protein phosphorylation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Jos Jonkers,et al.  Mice Deficient for All PIM Kinases Display Reduced Body Size and Impaired Responses to Hematopoietic Growth Factors , 2004, Molecular and Cellular Biology.

[37]  R. Aebersold,et al.  Identification of the Autophosphorylation Sites of theXenopus laevis Pim-1 Proto-oncogene-encoded Protein Kinase* , 1997, The Journal of Biological Chemistry.

[38]  Lewis C Cantley,et al.  A rapid method for determining protein kinase phosphorylation specificity , 2004, Nature Methods.

[39]  T. Möröy,et al.  The Oncogenic Serine/Threonine Kinase Pim-1 Phosphorylates and Inhibits the Activity of Cdc25C-associated Kinase 1 (C-TAK1) , 2004, Journal of Biological Chemistry.

[40]  A. Kraft,et al.  The PIM-2 Kinase Phosphorylates BAD on Serine 112 and Reverses BAD-induced Cell Death* , 2003, Journal of Biological Chemistry.

[41]  P. Hammerman,et al.  Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival. , 2005, Blood.

[42]  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.

[43]  K. Noguchi,et al.  Physical and Functional Interactions between Pim-1 Kinase and Cdc25A Phosphatase , 1999, The Journal of Biological Chemistry.

[44]  Shuichi Kaneko,et al.  Aberrant expression of serine/threonine kinase Pim‐3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines , 2005, International journal of cancer.

[45]  P. Laird,et al.  Impaired interleukin-3 response in Pim-1-deficient bone marrow-derived mast cells. , 1993, Blood.

[46]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[47]  Jun Li,et al.  Structural Basis of Constitutive Activity and a Unique Nucleotide Binding Mode of Human Pim-1 Kinase* , 2005, Journal of Biological Chemistry.

[48]  Brian A. Hemmings,et al.  Crystal structure of an activated Akt/Protein Kinase B ternary complex with GSK3-peptide and AMP-PNP , 2002, Nature Structural Biology.

[49]  Gouri Nanjangud,et al.  Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas , 2001, Nature.

[50]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[51]  A. Edelman,et al.  Similar substrate recognition motifs for mammalian AMP‐activated protein kinase, higher plant HMG‐CoA reductase kinase‐A, yeast SNF1, and mammalian calmodulin‐dependent protein kinase I , 1995, FEBS letters.

[52]  M. Fleming,et al.  Pim-1 Ligand-bound Structures Reveal the Mechanism of Serine/Threonine Kinase Inhibition by LY294002* , 2005, Journal of Biological Chemistry.

[53]  A. Berns,et al.  Evidence for the involvement of pim‐2, a new common proviral insertion site, in progression of lymphomas. , 1989, The EMBO journal.

[54]  J. Herrero,et al.  Exceptional Disfavor for Proline at the P+1 Position among AGC and CAMK Kinases Establishes Reciprocal Specificity between Them and the Proline-directed Kinases* , 2005, Journal of Biological Chemistry.

[55]  Zhou Songyang,et al.  Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes* , 1997, The Journal of Biological Chemistry.

[56]  Z. Wang,et al.  Pim-1: a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis. , 2001, Journal of veterinary science.

[57]  Wim Quint,et al.  Murine leukemia virus-induced T-cell lymphomagenesis: Integration of proviruses in a distinct chromosomal region , 1984, Cell.

[58]  K. Parang,et al.  Designing bisubstrate analog inhibitors for protein kinases. , 2002, Pharmacology & therapeutics.

[59]  P. Hammerman,et al.  Lymphocyte Transformation by Pim-2 Is Dependent on Nuclear Factor-κB Activation , 2004, Cancer Research.

[60]  T. Taira,et al.  Identification of heterochromatin protein 1 (HP1) as a phosphorylation target by Pim‐1 kinase and the effect of phosphorylation on the transcriptional repression function of HP1 1 , 2000, FEBS letters.

[61]  T. Graeber,et al.  Myc-driven murine prostate cancer shares molecular features with human prostate tumors. , 2003, Cancer cell.