Characterization of the proto-oncogene Pim-1: Kinase activity and substrate recognition sequence

Friedmann, Michael; Nissen, Mark S.; Hoover, Debra S.; Reeves, Raymond; Magnuson, Nancy S.

doi:10.1016/0003-9861(92)90454-5

Cited by 66 publications

(49 citation statements)

References 30 publications

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“…Likewise, we observed that human GST-Pim-1 also autophosphorylates on phosphotyrosine by phosphoamino acid analysis and Western blotting with anti-phosphotyrosine antibodies (data not shown). The autophosphorylation of Pim-1 on tyrosine was unexpected because the most recent studies have reported that the Pim-1 autophosphorylated strictly on serine and threonine residues (14,26,27,37).…”

Section: Resultsmentioning

confidence: 99%

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Identification of the Autophosphorylation Sites of theXenopus laevis Pim-1 Proto-oncogene-encoded Protein Kinase

Palaty

Kalmar

Tai

et al. 1997

Journal of Biological Chemistry

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Pim-1 is an oncogene-encoded serine/threonine kinase expressed primarily in cells of the hematopoietic and germ line lineages. Previously identified only in mammals, pim-1 cDNA was cloned and sequenced from the African clawed frog Xenopus laevis. The coding region of Xenopus pim-1 encoded a protein of 324 residues, which exhibited 64% amino acid identity with the full-length human cognate. Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and in COS cells. Phosphoamino acid analysis revealed that recombinant Pim-1 autophosphorylated on serine and threonine and to a more limited extent on tyrosine. Electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites, and the primary autophosphorylation site of GST-Pim-1 was identified as Ser-190 with Thr-205 and Ser-4 being minor sites. Ser-190, which immediately follows the high conserved Asp-Phe-Gly motif in catalytic subdomain VII, is also featured in more than 20 other protein kinases. To evaluate the importance of the Ser-190 site on the phosphotransferase activity of Pim-1, Ser-190 was mutated to either alanine or glutamic acid, and the constructs were expressed in bacteria as GST fusion proteins and in COS cells. These mutants confirmed that Ser-190 is a major autophosphorylation site of Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 residue may serve to activate this kinase.

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Section: Resultsmentioning

confidence: 99%

“…Nor has the phosphotransferase activity of the kinase been shown to be affected by phosphorylation. Pim-1 can autophosphorylate, but the sites of autophosphorylation and the functional consequences of this event have not yet been determined (14,(25)(26)(27).…”

mentioning

confidence: 99%

Identification of the Autophosphorylation Sites of theXenopus laevis Pim-1 Proto-oncogene-encoded Protein Kinase

Palaty

Kalmar

Tai

et al. 1997

Journal of Biological Chemistry

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“…However, the possibility that Pim-1 directly phosphorylates c-Myc and stimulates its activity as a transcriptional activator is unlikely based on the following lines of evidence. First, c-Myc does not contain the substrate recognition sequence for Pim-1 kinase, (Arg/Lys) 3 -X-Ser/Thr-X' (X' is likely neither a basic nor a large hydrophobic residue) (Friedmann et al, 1992). Indeed, according to Saris et al (1991), c-Myc protein was not phosphorylated by Pim-1 in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…For this purpose, we created a kinase-defective Pim-1 mutant (Pim-1K67M) by the conversion of Met at position 67 of the protein into Lys by site-directed mutagenesis. This Pim-1K67M mutant has been shown to lose its intrinsic kinase activity in in vitro kinase assays (Saris et al, 1991;Friedmann et al, 1992). We transfected pcDNA3pim-1K67M expressing this Pim-1 mutant into Rat-1/c-Myc#7 cells and isolated stable transfectants (Rat-1/c-Myc/Pim-1K67M).…”

Section: Pim-1 Stimulates Rather Than Inhibits C-myc-mediated Apoptosismentioning

confidence: 99%

Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation

Mochizuki¹,

Kitanaka²,

Noguchi³

et al. 1997

Oncogene

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Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that, similar to Bcl-2, Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, we examined the e ect of Pim-1 expression on c-Myc-mediated apoptosis of Rat-1 ®broblasts triggered by serum deprivation. Our results showed that, rather than inhibiting apoptosis, Pim-1 expression stimulated cMyc-mediated apoptosis in Rat-1 ®broblasts. Pim-1 stimulated c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a speci®c caspase inhibitor abolished the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant failed to stimulate c-Myc-mediated apoptosis, and Pim-1 expression alone in the absence of c-Myc overexpression did not induce apoptosis of serumdeprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway were required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 ®broblasts. Our results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) which regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation.

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“…They form hydrogen bonds with the catalytically essential Lys67 (Friedmann et al, 1992) and longer interactions with Asp186. All three compounds also form water-mediated inter- Interactions between compound 1 and Pim-1 kinase.…”

Section: Inhibitor-protein Interactionsmentioning

confidence: 99%

Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor

et al. 2012

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PDB References: Pim-1, complex with compound 1, 3umx; complex with compound 2, 4eny; complex with compound 3, 4enx.The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012), J. Med. Chem. 55, 5151-5156]. The report described the process of optimization of the structure-activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the 3 strand located above the binding site, is not usually observed in Pim-1 structures.

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Characterization of the proto-oncogene Pim-1: Kinase activity and substrate recognition sequence

Cited by 66 publications

References 30 publications

Identification of the Autophosphorylation Sites of theXenopus laevis Pim-1 Proto-oncogene-encoded Protein Kinase

Identification of the Autophosphorylation Sites of theXenopus laevis Pim-1 Proto-oncogene-encoded Protein Kinase

Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation

Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor

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