Pim-1 Ligand-bound Structures Reveal the Mechanism of Serine/Threonine Kinase Inhibition by LY294002
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m413155200
ISSN1083-351X
AutoresMarc Jacobs, James Black, Olga Futer, Lora Swenson, Brian Hare, Mark Fleming, Kumkum Saxena,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoPim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167–179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002·Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases. Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167–179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002·Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases. The Pim-1 oncogene was first identified as the preferred site for integration of the slow transforming Maloney murine leukemia virus in lymphoblastic T-cells (1.Wang Z. Bhattacharya N. Weaver M. Petersen K. Meyer M. Gapter L. Magnuson N.S. J. Vet. Sci. 2001; 2: 167-179Crossref PubMed Scopus (221) Google Scholar). Direct evidence for the oncogene potential of the Pim-1 gene comes from the study of transgenic mice in which overexpression of Pim-1 produces a low but spontaneous rate of tumor incidence (2.Domen J. van der Lugt N.M. Laird P.W. Saris C.J. Berns A. Leukemia. 1993; 7: S108-S112PubMed Google Scholar). These mice are highly susceptible to chemical carcinogens, x-ray radiation, and Maloney murine leukemia virus-induced lymphomagenesis. Pim-1 knock-out mice did not show any obvious phenotype, suggesting in vivo functional redundancy of this highly conserved oncogene (3.Domen J. van der Lugt N.M. Acton D. Laird P.W. Linders K. Berns A. J. Exp. Med. 1993; 178: 1665-1673Crossref PubMed Scopus (71) Google Scholar). Since the initial report of the cloning of mouse Pim-1 gene (4.Selten G. Cuypers H.T. Boelens W. Robanus-Maandag E. Verbeek J. Domen J. van Beveren C. Berns A. Cell. 1986; 46: 603-611Abstract Full Text PDF PubMed Scopus (143) Google Scholar), Pim-1 has been cloned from human, rat, bovine, and zebrafish cDNA libraries (1.Wang Z. Bhattacharya N. Weaver M. Petersen K. Meyer M. Gapter L. Magnuson N.S. J. Vet. Sci. 2001; 2: 167-179Crossref PubMed Scopus (221) Google Scholar). In humans, the Pim-1 gene is expressed mainly in the developing fetal liver and spleen (5.Amson R. Sigaux F. Przedborski S. Flandrin G. Givol D. Telerman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8857-8861Crossref PubMed Scopus (218) Google Scholar) and in hematopoietic malignancies (6.Meeker T.C. Nagarajan L. ar-Rushdi A. Rovera G. Huebner K. Croce C.M. Oncogene Res. 1987; 1: 87-101PubMed Google Scholar, 7.Nagarajan L. Louie E. Tsujimoto Y. ar-Rushdi A. Huebner K. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2556-2560Crossref PubMed Scopus (81) Google Scholar). Two homologs of the Pim-1 gene, pim-2 (8.van der Lugt N.M. Domen J. Verhoeven E. Linders K. van der Gulden H. Allen J. Berns A. EMBO J. 1995; 14: 2536-2544Crossref PubMed Scopus (170) Google Scholar) and pim-3/kid-1 (9.Feldman J.D. Vician L. Crispino M. Tocco G. Marcheselli V.L. Bazan N.G. Baudry M. Herschman H.R. J. Biol. Chem. 1998; 273: 16535-16543Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), have also been identified. The human Pim-1 gene encodes a 313-amino acid serine/threonine kinase (10.Padma R. Nagarajan L. Cancer Res. 1991; 51: 2486-2489PubMed Google Scholar) and is associated with multiple cellular functions such as proliferation, differentiation, apoptosis, and tumorigenesis (1.Wang Z. Bhattacharya N. Weaver M. Petersen K. Meyer M. Gapter L. Magnuson N.S. J. Vet. Sci. 2001; 2: 167-179Crossref PubMed Scopus (221) Google Scholar). Several cellular substrates of Pim-1 have been identified, including the transcription factors cMyb (11.Winn L.M. Lei W. Ness S.A. Cell Cycle. 2003; 2: 258-262Crossref PubMed Scopus (51) Google Scholar) and NFATc1 (12.Rainio E.M. Sandholm J. Koskinen P.J. J. Immunol. 2002; 168: 1524-1527Crossref PubMed Scopus (109) Google Scholar), the transcriptional co-activator of cMyb p100 (13.Leverson J.D. Koskinen P.J. Orrico F.C. Rainio E.M. Jalkanen K.J. Dash A.B. Eisenman R.N. Ness S.A. Mol. Cell. 1998; 2: 417-425Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), phosphatases Cdc25A (14.Mochizuki T. Kitanaka C. Noguchi K. Muramatsu T. Asai A. Kuchino Y. J. Biol. Chem. 1999; 274: 18659-18666Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) and PTPU2S (15.Wang Z. Bhattacharya N. Meyer M.K. Seimiya H. Tsuruo T. Tonani J.A. Magnuson N.S. Arch. Biochem. Biophys. 2001; 390: 9-18Crossref PubMed Scopus (45) Google Scholar), Pim-1-associated protein 1 (16.Maita H. Harada Y. Nagakubo D. Kitaura H. Ikeda M. Tamai K. Takahashi K. Ariga H. Iguchi-Ariga S.M. Eur. J. Biochem. 2000; 267: 5168-5178Crossref PubMed Scopus (55) Google Scholar), cell cycle inhibitor p21/WAF1 (17.Wang Z. Bhattacharya N. Mixter P.F. Wei W. Sedivy J. Magnuson N.S. Biochim. Biophys. Acta. 2002; 1593: 45-55Crossref PubMed Scopus (172) Google Scholar), heterochromatin protein 1 (18.Koike N. Maita H. Taira T. Ariga H. Iguchi-Ariga S.M. FEBS Lett. 2000; 467: 17-21Crossref PubMed Scopus (98) Google Scholar), TRAF2/SNX6 (19.Ishibashi Y. Maita H. Yano M. Koike N. Tamai K. Ariga H. Iguchi-Ariga S.M. FEBS Lett. 2001; 506: 33-38Crossref PubMed Scopus (44) Google Scholar), and nuclear mitotic apparatus (20.Bhattacharya N. Wang Z. Davitt C. McKenzie I.F. Xing P.X. Magnuson N.S. Chromosoma. 2002; 111: 80-95Crossref PubMed Scopus (94) Google Scholar). The consensus sequence for Pim-1 substrate recognition is Lys/Arg-Lys/Arg-Arg-Lys/Arg-Leu-Ser/Thr-Xaa, where Xaa is an amino acid with a small side chain (21.Friedmann M. Nissen M.S. Hoover D.S. Reeves R. Magnuson N.S. Arch. Biochem. Biophys. 1992; 298: 594-601Crossref PubMed Scopus (67) Google Scholar). The expression of Pim-1 is induced by a number cytokines, mitogens, and hormones (reviewed in Ref. 1.Wang Z. Bhattacharya N. Weaver M. Petersen K. Meyer M. Gapter L. Magnuson N.S. J. Vet. Sci. 2001; 2: 167-179Crossref PubMed Scopus (221) Google Scholar). The JAK/STAT (22.Nagata Y. Nagahisa H. Nagasawa T. Todokoro K. Leukemia. 1997; 11: 435-438PubMed Google Scholar, 23.Krumenacker J.S. Narang V.S. Buckley D.J. Buckley A.R. J. Neuroimmunol. 2001; 113: 249-259Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), AKT (24.Krishnan N. Pan H. Buckley D.J. Buckley A. Endocrine. 2003; 20: 123-130Crossref PubMed Scopus (18) Google Scholar), mitogen-activated protein kinase, and phosphatidylinositol 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; DTT, dithiothreitol; GST, glutathione S-transferase; PKA, cAMP-dependent kinase; AMP-PNP, adenosine 5′-(β,γ-iminotriphosphate); CK2, casein kinase 2. (23.Krumenacker J.S. Narang V.S. Buckley D.J. Buckley A.R. J. Neuroimmunol. 2001; 113: 249-259Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) pathways may all mediate Pim-1 expression. The x-ray structure of Pim-1 was pursued, in part, to determine how an unusual sequence feature in the active site affects ligand binding. In protein kinases, one hydrogen bond between the ATP (N1 atom) and a protein main chain NH is highly conserved. In the Pim-1 sequence, however, a proline (Pro123) occupies this position, so the main chain amide nitrogen is not available to participate in a hydrogen bond. A proline at this position is extremely rare; in fact, no other kinases with known structure have a similarly placed proline. Thus, the hydrogen bond to N1 of ATP is not necessary for substrate binding or catalysis in these kinases, and other interactions are sufficient to correctly position ATP. Qian et al. (25.Qian K.C. Wang L. Hickey E.R. Studts J. Barringer K. Penge C. Kronkaitis A. Li J. White A. Mische S. Farmer B. J. Biol. Chem. November 3, 2004; 280: 6130-6137Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) have determined the structures of both unliganded Pim-1 and the Pim-1·AMP-PNP complex. This work showed the mechanism by which Pim-1 is constitutively active and the manner in which an ATP analog is bound in the absence of the one of the main chain hydrogen bond donors. In our study, two kinase inhibitors, staurosporine and adenosine, both of which accept a hydrogen bond from this main chain NH in other kinase structures, were chosen for co-crystallization with Pim-1. The conformations of these ligands further elucidate the interactions needed for ligand positioning in the absence of a conserved hydrogen bond. In the course of determining the Pim-1 structure, similarities with the active site of PI3K were observed. The PI3K inhibitor, LY294002, was also found to inhibit Pim-1; we determined this additional co-complex structure to better understand the mechanisms by which LY294002 inhibits protein kinases. Cloning and Expression of Pim-1—Full-length Pim-1 (residues Met 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; DTT, dithiothreitol; GST, glutathione S-transferase; PKA, cAMP-dependent kinase; AMP-PNP, adenosine 5′-(β,γ-iminotriphosphate); CK2, casein kinase 2.–Lys313) was cloned in two parts by PCR from a human IMAGE Consortium clone (GenBank™ accession number GI 1845036) and from a human bone marrow cDNA library (BD Biosciences, Clontech, Palo Alto, CA). The pieces were fused by PCR and inserted into the NdeI and EcoRI sites of the dual promoter vector pBEV1, encoding a protein with an N-terminal His6 tag and thrombin cleavage site (26.Chambers S.P. Austen D.A. Fulghum J.R. Kim W.M. Protein Expression Purif. 2004; 36: 40-47Crossref PubMed Scopus (63) Google Scholar). The amino acid sequence of this Pim-1 clone is identical to SwissProt entry P11309. BL21/DE3 pLysS Escherichia coli cells were transformed with the construct encoding full-length human Pim-1 kinase, using a standard transformation protocol (Stratagene, La Jolla, CA). Freshly transformed cells were grown at 37 °C in brain heart infusion medium (DIFCO Laboratories, Detroit, MI) supplemented with 100 μg/ml carbenicillin and 35 μg/ml chloramphenicol. The cells were grown at 37 °C up to an optical density of 0.75 at 600 nm, and expression was induced at 28 °C with 1 mm isopropyl β-d-thiogalactopyranoside. The cells were harvested via centrifugation 4 h post-induction and stored at –80 °C prior to purification. Protein Purification—Frozen cell pellets (∼30 g) were thawed in 7 volumes of Buffer A (50 mm HEPES, pH 7.8, 300 mm NaCl, 10% (v/v) glycerol, 3 mm β-mercaptoethanol) containing 0.1% (v/v) Tween 20, 50 μm diisopropyl fluorophosphate, 1 μg/ml E-64, 1 μg/ml leupeptin, and 10 μg/ml pepstatin (Roche Applied Science) and lysed in a microfluidizer (Microfluidics, Newton, MA). The lysate was centrifuged at 54,000 × g for 45 min, and the supernatant was incubated with 1 ml of Talon metal affinity resin (BD Biosciences, Clontech)/5 mg of protein overnight at 4 °C. The resin was washed with 20 column volumes of Buffer A and eluted with Buffer A containing 100 mm imidazole. Fractions containing Pim-1 were pooled and concentrated by ultrafiltration using a 30-kDa molecular mass cut-off membrane in an Amicon stirred cell concentrator (Millipore, Billerica, MA). The Pim-1 fraction was loaded onto a Superdex 200 column (90 × 2.6 cm; Amersham Biosciences) equilibrated in Buffer B (50 mm HEPES, pH 7.8, 200 mm NaCl, 10% (v/v) glycerol, and 5 mm β-mercaptoethanol). The fractions were pooled based on SDS-PAGE, diluted to 25 mm NaCl with 50 mm HEPES, pH 7.8, 10% (v/v) glycerol, and 5 mm dithiothreitol (DTT), and loaded onto a 8-ml prepacked MonoQ (HR 10/10) anion exchange column (Amersham Biosciences) equilibrated in Buffer C (50 mm HEPES, pH 7.8, 20 mm NaCl, 10% (v/v) glycerol, 5 mm DTT). Pim-1 was eluted using a gradient of 0–40% Buffer D (buffer C plus 1 m NaCl) over 60 column volumes. Peak fractions were collected as four separate pools (I–IV) based on the elution chromatogram. The Pim-1 fraction was dialyzed into 20 mm Tris, pH 8.0 (25 °C), 100 mm NaCl, 5 mm DTT and concentrated to 10 mg/ml using a 10-kDa molecular mass cut-off Vivaspin concentrator (Vivascience, Hanover, Germany). The identity of the purified Pim-1 was confirmed by N-terminal amino acid sequencing. Typically, preparations contained a mixture of species with 0–5 phosphoryl groups, which were partially resolved by anion exchange chromatography (Fig. 1). Phosphoamino acid analysis showed that Pim-1 purified from E. coli was extensively phosphorylated in the His6 tag (MGSSHHHHHHSSGLVPRGSH), and the four MonoQ pools differed mainly in the degree of phosphorylation in this region. Dephosphorylation of Pim-1 with Lambda phosphatase (New England Biolabs) followed by autophosphorylation showed that Pim-1 readily autophosphorylates in the His6 tag region (data not shown). Ser261 was the major phosphorylation site observed in Pools III and IV. Other minor sites, Ser8, Thr23, and Ser98, were phosphorylated to varying degrees in each pool. Pim-1 crystallized from different MonoQ pools yielded similar crystal forms. The phosphorylation state of each of the MonoQ-purified pools I-IV of Pim-1 was determined by electrospray mass spectrometry of thrombin-cleaved Pim-1. Spectra were collected using a Micromass Quattro II triple quadrupole mass spectrometer (Waters Corp., Milford, MA) (27.Fox T. Fitzgibbon M.J. Fleming M.A. Hsiao H.M. Brummel C.L. Su M.S. FEBS Lett. 1999; 461: 323-328Crossref PubMed Scopus (29) Google Scholar). Phosphorylation sites were identified from tryptic digests of the MonoQ-purified pools I–IV to liquid chromatography-tandem mass spectrometry on a QSTAR Pulsar quadrupole time-of-flight tandem mass spectrometer (AB/MDS-Sciex, Toronto, Canada) equipped with a nanoelectrospray ion source (MDS Protana, Odese, Denmark). The data were analyzed using the Mascot search engine (Matrix Science, London, UK). Kinase Assays—A coupled-enzyme assay (28.Fox T. Coll J.T. Xie X. Ford P.J. Germann U.A. Porter M.D. Pazhanisamy S. Fleming M.A. Galullo V. Su M.S. Wilson K.P. Protein Sci. 1998; 7: 2249-2255Crossref PubMed Scopus (130) Google Scholar) was used to quantify the ADP generated in the kinase reaction with S6 peptide (RRRLSSLRA) (American Peptide Co, Sunnyvale, CA) as the phosphoacceptor substrate. The assay was carried out in a total volume of 100 μl in 0.1 m HEPES, pH 7.6, containing 10 mm MgCl2, 2.5 mm phosphoenolpyruvate, 0.2 mm NADH, 30 μg/ml pyruvate kinase, 10 μg/ml lactate dehydrogenase (Roche Applied Science), and 2 mm DTT in a 96-well plate and read at 340 nm at 30 °C on a Spectramax spectrophotometer (Molecular Devices, Sunnyvale, CA). Pim-1 concentration was 25 nm in all assays. The reaction was started by the addition of ATP after 10 min of preincubation of the reaction mixture at 30 °C. Substrate concentrations were 1 mm S6 peptide, 2 mm ATP for activity assays and 40 μm S6 peptide, 100 μm ATP for IC50 determinations. Inhibitors were dissolved in Me2SO and added to the reaction to 2.5% Me2SO final at the beginning of preincubation period. Kinetic analysis was performed by nonlinear regression fitting using the program Prism (GraphPad Software, San Diego, CA) (Table I).Table IIC50 values of kinase inhibitors determined for Pim-1 Open table in a new tab Crystallization and X-ray Analysis—Pim-1 crystals were grown by the vapor diffusion method at 22 °C. Equal volumes of protein (12 mg/ml protein, 20 mm HEPES, pH 8.0, 100 mm NaCl, 5 mm DTT) and well solution (1 m (NH4)2HPO4, 100 mm citrate buffer pH 5.5, 200 mm NaCl) were mixed and suspended over 1 ml of well solution. Over 4 days, the crystals reached a final size of ∼250 × 40 × 40 μm. Crystals were harvested and flash-frozen in a solution composed of the well solution with 30% (v/v) glycerol. A complex of Pim-1 with either staurosporine (Sigma-Aldrich) or the inhibitor LY294002 (Calbiochem, La Jolla, CA) was made by soaking unliganded crystals (grown as above) with 500 μm compound and 5% Me2SO (final concentration) for 24 h at room temperature. The adenosine·Pim-1 complex was made by adding adenosine (2 mm) to the protein prior to crystallization. For the staurosporine and LY294002 complexes, x-ray diffraction data were recorded using a RU-200 x-ray generator and RaxisV++ detector (Rigaku, The Woodlands, TX), and the intensities were integrated and scaled using CrystalClear (29.CrystalClearAn Integrated Program for the Collection and Processing of Area Detector Data. Rigaku/MSC, The Woodlands, TX1997–2002Google Scholar). Diffraction data for the adenosine complex crystals were recorded at Beamline 5.0.2 at the Advanced Light Source (Lawrence Berkeley Laboratories, Berkeley, CA). The intensities were integrated and scaled using the programs DENZO and SCALEPACK (30.Otwinoski Z. L. Sawyer N.W.I. Bailey S. Oscillation Data Reduction Program: Data Collection and Processing. SERC Daresbury Laboratory, Warrington, UK1993Google Scholar) and CrystalClear. The structure was determined by molecular replacement using homology models based upon phosphorylase kinase (Protein Data Bank code 1PHK) (31.Owen D.J. Noble M.E. Garman E.F. Papageorgiou A.C. Johnson L.N. Structure. 1995; 3: 467-482Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and death-associated protein kinase (Protein Data Bank code 1JKK) (32.Tereshko V. Teplova M. Brunzelle J. Watterson D.M. Egli M. Nat. Struct. Biol. 2001; 8: 899-907Crossref PubMed Scopus (91) Google Scholar). The molecular replacement solution was determined using AMORE (Navaza, CCP4 distribution) (33.CCP4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar). The crystals belong to the space group P65, and a single protein monomer comprises the asymmetric unit. The protein model was built using QUANTA (Accelrys, San Diego, CA) and refined with both CNX (Accelrys) (34.Rice L.M. Brunger A.T. Proteins. 1994; 19: 277-290Crossref PubMed Scopus (382) Google Scholar) and BUSTER (Global Phasing Inc., Cambridge, UK) (35.Roversi P. Blanc E. Vonrhein C. Evans G. Bricogne G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1316-1323Crossref PubMed Scopus (69) Google Scholar) (Table II).Table IIData collection and refinement statisticsData setStaurosporineAdenosineLY294002Data collection X-ray sourceRigaku RU-H3RALS 5.0.2Rigaku RU-H3R Space groupP65P65P65 Unit cell parameters (Å)a = b = 97.7a = b = 98.3a = b = 97.7c = 80.5c = 80.4c = 80.7 Resolution (Å)20-2.1520-2.420-2.5 Unique reflections226151643014445 Redundancy3.65.23.1 Completeness (%)aThe values for the highest resolution shell are shown in parentheses.94.9 (74.8)94.3 (96.1)94.9 (87.6) Rmerge aThe values for the highest resolution shell are shown in parentheses.,bRmerge = Σhkl Σi |I(hkl)i - 〈I(hkl〉|/Σhkl Σi〈I(hkl)i〉 over i observations of reflection hkl. R factor = Σ∥Fobs| - |Fcalc∥/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Free R factor is calculated from a randomly chosen subset of reflections not used for refinement.0.050 (0.250)0.060 (0.361)0.072 (0.336) 〈I/σaThe values for the highest resolution shell are shown in parentheses.〉10.6 (2.3)14.7 (3.9)12.0 (2.6)Refinement Reflections used225261615214206 Test reflections170612681097 R factor0.2050.2100.208 Free R factor (% data)0.233 (7.6)0.246 (7.9)0.259 (7.7) Root mean square deviation Bond lengths (Å)0.0150.0070.009 Bond angles (°)1.71.31.2 Dihedral angles (°)23.122.822.2 Protein atoms220222022202 Solvent atoms14281136a The values for the highest resolution shell are shown in parentheses.b Rmerge = Σhkl Σi |I(hkl)i - 〈I(hkl〉|/Σhkl Σi〈I(hkl)i〉 over i observations of reflection hkl. R factor = Σ∥Fobs| - |Fcalc∥/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Free R factor is calculated from a randomly chosen subset of reflections not used for refinement. Open table in a new tab The refined model consists of the protein kinase catalytic domain. Although full-length protein was used for crystallization (313 residues), 32 residues at the N terminus, 8 residues at the C terminus, and 4 residues in one loop (80–83) were not sufficiently ordered to be built into the electron density. Phosphorylation of Ser261 is clearly visible in the electron density map. The phosphoserine side chain participates in both intra- and intermolecular interactions and may be important in formation of the crystal packing interactions. Also, the electron density map reveals additional density adjacent to the sulfur of Cys161, indicating an adduct at this residue. The electron density was large enough to accommodate four nonhydrogen atoms; it was modeled as a β-mercaptoethanol adduct; however, it is also consistent with a partially ordered DTT adduct. Both DTT and β-mercaptoethanol were used in the purification. Enzymatic Activity—Kinase activity assays of the MonoQ pools I–IV were performed to determine whether the observed phosphorylation affected the catalytic activity. All four pools showed very similar kinetic parameters (kcat = 4 ± 0.4 s–1, peptide Km = 51 ± 2 μm, and ATP Km = 120 ± 16 μm). Pim-1 purified from E. coli was phosphorylated at Ser261 as well as multiple sites in the His tag region. Palaty et al. (36.Palaty C.K. Kalmar G. Tai G. Oh S. Amankawa L. Affolter M. Aebersold R. Pelech S.L. J. Biol. Chem. 1997; 272: 10514-10521Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) have identified Ser190 in Xenopus Pim-3 as the major autophosphorylation site and showed that S190A and S190E mutants are 7-fold less active than the wild type Pim-3. The equivalent residue in human Pim-1, Ser189, was not phosphorylated in the E. coli purified preparations. The fact that all four MonoQ Pim-1 pools exhibit very similar kinetic parameters indicates that the enzyme is constitutively active and that the phosphorylation state does not affect enzymatic activity. The specific activity (5 ± 0.2 μmol/min/mg) observed here is much higher than previously reported (21.Friedmann M. Nissen M.S. Hoover D.S. Reeves R. Magnuson N.S. Arch. Biochem. Biophys. 1992; 298: 594-601Crossref PubMed Scopus (67) Google Scholar, 36.Palaty C.K. Kalmar G. Tai G. Oh S. Amankawa L. Affolter M. Aebersold R. Pelech S.L. J. Biol. Chem. 1997; 272: 10514-10521Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 37.Hoover D. Friedmann M. Reeves R. Magnuson N.S. J. Biol. Chem. 1991; 266: 14018-14023Abstract Full Text PDF PubMed Google Scholar, 38.Palaty C.K. Clark-Lewis I. Leung D. Pelech S.L. Biochem. Cell Biol. 1997; 75: 153-162Crossref PubMed Scopus (48) Google Scholar). It is 60-fold greater than that reported by Friedman et al. (21.Friedmann M. Nissen M.S. Hoover D.S. Reeves R. Magnuson N.S. Arch. Biochem. Biophys. 1992; 298: 594-601Crossref PubMed Scopus (67) Google Scholar) for human GST-Pim-1 using a histone H1 peptide (KRRASGP) and over 104-fold greater than that reported by Palaty et al. (38.Palaty C.K. Clark-Lewis I. Leung D. Pelech S.L. Biochem. Cell Biol. 1997; 75: 153-162Crossref PubMed Scopus (48) Google Scholar) for GST fusions of human Pim-1 using S6 peptide (AKRRRLSSLRA). Because both studies utilized GST fusions for expression and purification, it is possible that this large protein tag had a detrimental effect on enzyme activity, either by interfering with substrate access to the active site or with overall protein folding. A panel of kinase inhibitors was evaluated for their ability to inhibit Pim-1. Staurosporine and structurally similar compounds, such as K-252a and bisindolyl-maleimides I and IX, were found to inhibit Pim-1 with submicromolar potency (Table I). These compounds are nonspecific inhibitors of Ser/Thr and Tyr kinases (39.Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3944) Google Scholar). Surprisingly, LY294002 was found to be an inhibitor of Pim-1 with IC50 = 4 μm. This compound was originally described as a specific inhibitor of PI3K with 1.4 μm IC50 (40.Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). Later, Davies et al. (39.Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3944) Google Scholar) reported that LY294002 inhibits PI3K and casein kinase 2 with a similar potency (10 and 6.9 μm, respectively). Protein Structure—The structure of Pim-1 has a global fold typical of protein serine/threonine kinases, consisting of two domains linked by a hinge region (Fig. 2). The smaller, N-terminal domain (residues 33–121) consists primarily of β-strands with one α-helix, and the C-terminal domain (residues 128–305) is largely α-helical. The active site is formed by a groove at the interface between these two domains and is enclosed by the hinge region (residues 122–127), the glycine rich loop (residues 44–52), and the activation loop (residues 186–210). The Pim-1 structure was compared with several other protein kinases with high sequence homology such as cAMP-dependent kinase (PKA) and phosphorylase kinase. When secondary structural elements are aligned, a root mean square deviation of 1.3 Å for C-α atom positions is observed between Pim-1 and both PKA or phosphorylase kinase (using 213 residues from phosphorylase kinase (Protein Data Bank code 1PHK) (31.Owen D.J. Noble M.E. Garman E.F. Papageorgiou A.C. Johnson L.N. Structure. 1995; 3: 467-482Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and 220 residues from cAMP-dependent kinase (Protein Data Bank code 1ATP) (41.Zheng J. Knighton D.R. ten Eyck L.F. Karlsson R. Xuong N. Taylor S.S. Sowadski J.M. Biochemistry. 1993; 32: 2154-2161Crossref PubMed Scopus (513) Google Scholar), respectively). The Pim-1 protein structures described here are similar to unliganded Pim-1 (25.Qian K.C. Wang L. Hickey E.R. Studts J. Barringer K. Penge C. Kronkaitis A. Li J. White A. Mische S. Farmer B. J. Biol. Chem. November 3, 2004; 280: 6130-6137Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), with a root mean square deviation of 0.4 Å for C-α atoms. The conformation of the glycine-rich loop (residues 45–52) in this structure differs from that of the PKA structures and most closely resembles that of unliganded Pim-1 (25.Qian K.C. Wang L. Hickey E.R. Studts J. Barringer K. Penge C. Kronkaitis A. Li J. White A. Mische S. Farmer B. J. Biol. Chem. November 3, 2004; 280: 6130-6137Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The Pim-1 glycine-rich loop moves toward the C-terminal domain, and Phe49 adopts a rotamer in which the side chain points toward the hinge region, thereby filling the space usually occupied by ATP phosphates (Fig. 2). In the Pim-1·AMP-PNP complex structure, the ligand phosphates displace Phe49, and the loop adopts a more open conformation. This is similar to GSK-3β, for instance, where the corresponding phenylalanine residue is observed both within the active site pointing toward the hinge (peptide complex) (42.Bax B. Carter P.S. Lewis C. Guy A.R. Bridges A. Tanner R. Pettman G. Mannix C. Culbert A.A. Brown M.J. Smith D.G. Reith A.D. Structure (Camb.). 2001; 9: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) and, in another structure (unliganded), outside the active site, pointing away from the hinge (43.ter Haar E. Coll J.T. Austen D.A. Hsiao H.M. Swenson L. Jain J. Nat. Struct. Biol. 2001; 8: 593-596Crossref PubMed Scopus (326) Google Scholar). The Pim-1 hinge sequence is unusual because of a two-residue insertion relative to kinases such as CDK-2 (44.De Bondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (833) Google Scholar) and JNK-3 (45.Xie X. Gu Y. Fox T. Coll J.T. Fleming M.A. Markland W. Caron P.R. Wilson K.P. Su M.S. Structure. 1998; 6: 983-991Abstract Full Text Full Text PDF PubMed Scopus
Referência(s)