Pim-1 Ligand-bound Structures Reveal the Mechanism of Serine/Threonine Kinase Inhibition by LY294002
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. 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 with and The cells were grown at 37 °C to an of at and expression was induced at °C with 1 The cells were and at °C to Protein cell were in of A 1 1 and and in a The was at for and the was with 1 of (BD Biosciences, of protein at The was with of A and with A Pim-1 were and by using a in an cell The Pim-1 was a in and The were to with and and a in Pim-1 was using a of 1 were as the The Pim-1 was into and to using a The of the Pim-1 was by N-terminal amino acid a of with which were by acid showed that Pim-1 from E. coli was in the His6 tag and the mainly in the of in this of Pim-1 with by showed that Pim-1 in the His6 tag region not was the site in and and were to in Pim-1 from crystal The of of the of Pim-1 was determined by of Pim-1. were using a T. M.S. FEBS Lett. 1999; PubMed Scopus Google Scholar). sites were identified from of the to a with a The were using the T. P.J. S. M.S. Protein Sci. 1998; 7: PubMed Scopus Google Scholar) was used to the in the kinase with as the The was in a of in kinase, and in a and at at °C a CA). Pim-1 was in all The was by the of ATP of of the at were 1 ATP for and ATP for were in and to the to at the of was by using the of kinase determined for Pim-1 in a new and were grown by the at of protein and were and 1 of the a of were and in a of the with A complex of Pim-1 with staurosporine or the inhibitor LY294002 La Jolla, was by unliganded as with and for at The complex was by adenosine to the protein to the staurosporine and LY294002 x-ray were using a x-ray and The and the were and using for the and of The Scholar). for the adenosine complex were at at the CA). The were and using the and Z. L. S. and Scholar) and The structure was determined by using kinase D.J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and protein kinase M. J. M. Biol. 2001; PubMed Scopus Google Scholar). The was determined using Biol. PubMed Scopus Google Scholar). The to the and a protein the The protein was using and with both L.M. PubMed Scopus Google Scholar) and E. C. G. G. Biol. 2000; PubMed Scopus Google Scholar) and cell for the are in for the are in of where and are the and structure is from a chosen of not used for for the are in Protein The for the are in of where and are the and structure is from a chosen of not used for in a new The of the protein kinase Although full-length protein was used for at the at the and in one were not to be into the of is in the The side chain in both and interactions and may be in of the crystal the reveals additional to the of an at this The was to it was as a however, it is also with a and were used in the of the were to determine the the showed and ATP Pim-1 from E. coli was at as as multiple sites in the tag et al. G. G. S. L. M. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) have identified in as the site and showed that and are active than the The residue in human Pim-1, was not in the E. coli The that all Pim-1 that the is constitutively active and that the does not The is than (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, G. G. S. L. M. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. Reeves R. Magnuson N.S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, D. Biochem. Cell Biol. 1997; PubMed Scopus Google Scholar). is than that by 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 using a and than that by et al. D. Biochem. Cell Biol. 1997; PubMed Scopus Google Scholar) for of human Pim-1 using both for expression and it is that this protein tag a by with substrate to the active site or with protein A of kinase was for to inhibit Pim-1. and such as and and were found to inhibit Pim-1 with These are of Ser/Thr and kinases S.P. H. M. Biochem. J. 2000; PubMed Scopus Google Scholar). LY294002 was found to be an inhibitor of Pim-1 with This was originally as a inhibitor of PI3K with C.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). et al. S.P. H. M. Biochem. J. 2000; PubMed Scopus Google Scholar) that LY294002 inhibits PI3K and casein kinase with a and Protein structure of Pim-1 has a fold typical of protein serine/threonine kinases, of two by a hinge region The N-terminal (residues primarily of with one and the (residues is The active site is by a at the between these two and is by the hinge region (residues the (residues and the (residues The Pim-1 structure was with other protein kinases with sequence such as cAMP-dependent kinase and are a of for is between Pim-1 and both or kinase from kinase D.J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and from cAMP-dependent kinase J. R. N. 1993; PubMed Scopus Google Scholar), The Pim-1 protein structures are 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 of for The of the (residues in this structure from that of the structures and 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 the and a in which the side chain the hinge the by ATP In the Pim-1·AMP-PNP complex structure, the ligand and the a more This is to for where the residue is both the active site the hinge B. C. A.R. A. R. G. C. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) in structure the active away from the hinge E. Austen D.A. L. J. Biol. 2001; PubMed Scopus Google Scholar). The Pim-1 hinge sequence is unusual of a two-residue insertion to kinases such as J. J. Kim 1993; PubMed Scopus Google Scholar) and Y. T. W. M.S. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and a residue insertion to A of the hinge of Pim-1 and is in and the insertion however, at the of insertion the hinge away from the ATP-binding site by to of the additional by the in main chain position is by the side which is the ATP-binding and with This hinge be for the of Pim-1 and a for at the position to of ATP. interactions with the of or with the side chain of be to kinase this two-residue insertion is PI3K C. L. 1999; PubMed Scopus Google Scholar). Although is sequence in the hinge region between PI3K and Pim-1, the main chain conformations are The PI3K and Pim-1 hinge at in interactions with the hinge with of Pim-1 and PI3K bound to adenosine, and ATP were to the of to the hinge The Pim-1 structure is by and in and PI3K is and in and and and of staurosporine and adenosine bound to Pim-1 are to in other protein kinase complex are between from the the and the In one hydrogen bond between the ligand and the hinge is observed. In the a hydrogen bond is between the main chain and the amino The nitrogen of staurosporine a hydrogen bond. The of proline at position of the two from the hydrogen bond to the The Pim-1 ligand structures were and with to the complex N. S. S. N. 1997; 36: PubMed Scopus Google Scholar), the adenosine in Pim-1 by the hinge into the additional by the proline insertion to the of the The of adenosine in the Pim-1 and structures are The position of the adenosine in the two structures in part, by the of the side chain to which the a hydrogen bond in Pim-1 and in the of staurosporine in Pim-1 and L. A. R. D. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) are in Pim-1 the hinge by and also a hydrogen bond to A of the ligand in the Pim-1 and the PI3K and C. L. 1999; PubMed Scopus Google Scholar, S. S. L. Cell. 2000; Full Text Full Text PDF PubMed Google Scholar) reveals a and a In PI3K, two hydrogen are between the main chain NH and and the ligands N1 and and staurosporine and and staurosporine bound to PI3K are the of the hinge by to the Pim-1 structure the staurosporine is an to the main chain of the hinge and such that the the the in the Pim-1 structure Although the hinge conformations between Pim-1 and PI3K are the of adenosine and staurosporine In fact, the of the ligands in Pim-1 more closely found in other protein kinases. The PI3K binding by the the hinge and the is in Pim-1. The of the of and the side chain at position of the the The the is by the side chain of in Pim-1. In the absence of the conserved of hydrogen to the a number of van der Waals the position of the LY294002 structure of PI3K inhibitor LY294002 bound to Pim-1 was the of the of the in the Pim-1 in as as the between the Pim-1 and PI3K bound to PI3K, the of LY294002 a hydrogen bond from the amide nitrogen of the as with N1 of ATP The structure of the complex reveals that the is to the PI3K structure, the LY294002 the bond to the two in the In this the with the hinge is between the main chain of and two of the The a hydrogen bond to a which in with the main chain amide of The of LY294002 the side of and the with in the The between the Pim-1 hinge and LY294002 are highly protein kinase ligands with the hinge hydrogen where the hydrogen is to or In this to with the these interactions are for LY294002 we the of the to be for hydrogen The to is and the between the and the LY294002 is and 2002; PubMed Scopus Google Scholar). The is between and The with LY294002 in Pim-1 are and the hydrogen and the amide bond be which is the in the complex. is that a of hydrogen are between LY294002 and the Pim-1 The that LY294002 to Pim-1, a protein with a to be the is in the two Although the have of the in to the binding of In fact, of the two reveals that the PI3K binding mode is by in Pim-1. the Pim-1 binding mode is with the PI3K in PI3K the and the of LY294002 but with the the bound in the Pim-1 The LY294002 is used to the of PI3K in cell and does not inhibit protein kinases S.P. H. M. Biochem. J. 2000; PubMed Scopus Google Scholar). is by in the of LY294002, so we not the structure of to LY294002 binding. both the Pim-1 and PI3K binding are by and in kinase by LY294002 is casein kinase The structures of and Pim-1 were to how LY294002 to The PI3K binding mode is by the side chain of in the active site LY294002 in the Pim-1 binding with a to a with in to kinases, LY294002 has also been to to with and In these by LY294002, but not by PI3K was and a LY294002 has been to in cells Cell 2002; PubMed Scopus Google Scholar), the and W. Wang J. Wang J. 2003; PubMed Scopus Google Scholar), and also to and inhibit J. R. A. Mol. Cell. 2003; PubMed Scopus Google Scholar). This may be to the that LY294002 is a and with hydrogen is that are other as of this and LY294002 be used with in cellular protein kinases, the hinge and the hydrogen to ATP are highly conserved. The Pim-1 structure reveals how a substrate complex the hinge is unusual in both sequence and The structures of the adenosine and staurosporine show van der Waals the as a conserved hydrogen bond to position the Although the Pim-1 hinge closely resembles the region in the active site of PI3K, the LY294002 with the of the two in This new binding mode a to show how LY294002 inhibit other protein kinases, and this structure be used to in the of inhibitors, of the Pim-1 active H. for of J. Fulghum for protein M. for for and G. and the at at the for in also J. S. and E. for