Structural Basis of Inhibitor Specificity of the Human Protooncogene Proviral Insertion Site in Moloney Murine Leukemia Virus (PIM-1) Kinase

Bullock, Alex N.; Debreczeni, J.; Fedorov, O.; Nelson, Adam; Knapp, Stefan

doi:10.1021/jm0504858

Cited by 144 publications

(147 citation statements)

References 54 publications

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“…This simple and cost-effective screening method allowed us to target both active and inactive kinases and has been shown to provide data that are consistent with orthogonal methods such as isothermal titration calorimetry and enzymatic assays (13,(15)(16)(17). A comparison of T m shift data with IC 50 values of two targets has been included in SI Fig.…”

Section: Resultsmentioning

confidence: 84%

“…The screening data were generated by using a thermal shift assay in which the kinase is stabilized upon inhibitor binding (13,14). This simple and cost-effective screening method allowed us to target both active and inactive kinases and has been shown to provide data that are consistent with orthogonal methods such as isothermal titration calorimetry and enzymatic assays (13,(15)(16)(17).…”

Section: Resultsmentioning

confidence: 99%

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A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases

Fedorov

Marsden

Pogačić

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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336

323

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Protein kinases play a pivotal role in cell signaling, and dysregulation of many kinases has been linked to disease development. A large number of kinase inhibitors are therefore currently under investigation in clinical trials, and so far seven inhibitors have been approved as anti-cancer drugs. In addition, kinase inhibitors are widely used as specific probes to study cell signaling, but systematic studies describing selectivity of these reagents across a panel of diverse kinases are largely lacking. Here we evaluated the specificity of 156 validated kinase inhibitors, including inhibitors used in clinical trials, against 60 human Ser/Thr kinases using a thermal stability shift assay. Our analysis revealed many unexpected cross-reactivities for inhibitors thought to be specific for certain targets. We also found that certain combinations of activesite residues in the ATP-binding site correlated with the detected ligand promiscuity and that some kinases are highly sensitive to inhibition using diverse chemotypes, suggesting them as preferred intervention points. Our results uncovered also inhibitor crossreactivities that may lead to alternate clinical applications. For example, LY333531, a PKC␤ inhibitor currently in phase III clinical trials, efficiently inhibited PIM1 kinase in our screen, a suggested target for treatment of leukemia. We determined the binding mode of this inhibitor by x-ray crystallography and in addition showed that LY333531 induced cell death and significantly suppressed growth of leukemic cells from acute myeloid leukemia patients.acute myeloid leukemia ͉ inhibitor selectivity ͉ LY333Ј531 ͉ PIM1 ͉ screening data

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Section: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases

Fedorov

Marsden

Pogačić

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

336

323

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“…In this assay, binding affinities are measured indirectly as a function of melting temperature increase (⌬T m ). Several studies have shown that ⌬T m correlates linearly with binding constants of ligands (18). Nucleotides demonstrated no interaction with haspin in the absence of divalent ions (Fig.…”

Section: Resultsmentioning

confidence: 91%

Structure and functional characterization of the atypical human kinase haspin

Eswaran

Patnaik

Filippakopoulos

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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167

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The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP ␥-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.ATP binding ͉ histone modification ͉ phosphorylation mechanism ͉ germ cell-specific gene 2 (Gsg2) ͉ mitosis E ukaryotic protein kinases (ePKs) constitute a large group of enzymes that regulate a vast diversity of cellular processes. Kinase activity depends on a number of highly conserved sequence motifs required for ATP/Mg 2ϩ binding and catalysis. About 10% of human ePKs lack one or more essential catalytic motifs and have been initially classified as inactive pseudokinases (1). However, a number of such proteins are active kinases, including haspin [haploid germ cell-specific nuclear protein kinase, encoded by Germ cell-specific gene 2 (Gsg2)]. Haspin lacks both the conserved ATP/Mg 2ϩ binding motif Asp-Phe-Gly (DFG), which is replaced by Asp-Tyr-Thr (DYT), and the Ala-Pro-Glu (APE) motif usually found at the C terminus of the activation segment (2). In addition, haspin shares only weak sequence homology with ePKs and contains a highly divergent kinase domain with several unique inserts (2, 3). Haspin mRNA is abundant in testis and also present in somatic cells (4, 5), and orthologues are found throughout eukaryotic phyla (2). Ectopically expressed haspin localizes to the nucleus in interphase and to the chromosomes in mitosis (4, 6), while depletion of haspin leads to premature chromatid separation, failure of chromosome alignment, and a block in mitosis in a prometaphase-like state (6, 7).Haspin autophosphorylates in vitro (4, 6, 8) and phosphorylates its only currently known substrate, histone H3, at threonine-3 (H3T3ph) both in vitro and in cells (6). H3T3 phosphorylation occurs specifically during mitosis and is particularly prominent at inner centromere regions (6, 7, 9). Recently, H3T3ph has been suggested to relieve an inhibitory effect of ...

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“…Indeed, small molecule inhibitors of the Pim-1 kinase have been recently described in vitro and in cell-based systems. [33][34][35] Targeting the Pim-1 kinase may be a beneficial addition to a traditional cancer chemotherapy regimen.…”

Section: Discussionmentioning

confidence: 99%

Pim-1 controls NF-κB signalling by stabilizing RelA/p65

et al. 2009

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Post-translational modification and degradation of proteins by the ubiquitin-proteasome system are key regulatory mechanisms in cellular responses to various stimuli. The NF-jB signaling pathway is controlled by the ubiquitin-mediated proteolysis. RelA/p65, which is a main subunit of NF-jB, is ubiquitinated for degradation by SOCS-1, but the functional mechanism of its ubiquitination remains poorly understood. In this study we show that phosphorylation of RelA/p65 at Ser276 prevents its degradation by ubiquitin-mediated proteolysis. In contrast, impairment of Ser276 phosphorylation affects constitutive degradation of RelA/p65. Importantly, we identify Pim-1 as a further kinase responsible for the phosphorylation of RelA/p65 at Ser276. Depletion of Pim-1 hinders not only Ser276 phosphorylation but also transactivation of RelA/p65 target genes. We also show that Pim-1 contributes to recruitment of RelA/p65 to jB-elements to activate NF-jB signalling after TNF-a stimulation. In concert with these results, the knockdown of Pim-1 impairs IL-6 production and augments apoptosis by interfering RelA/p65 activation. These findings provide a model in which Pim-1 phosphorylation of RelA/p65 at Ser276 allows defense against ubiquitin-mediated degradation and whereby exerts activation of NF-jB signalling. NF-kB is an inducible transcription factor that controls the expression of a number of proteins involved in the regulation of cell survival and immune response. 1 It is a dimmer formed from a multisubset family consisting of RelA/p65, RelB, c-Rel, p105/p50 (NF-kB1), and p100/p52 (NF-kB2). It is activated by a bewildering array of stimuli and its activation is regulated by multiple distinct signalling cascades, including inhibitors of the NF-kB (IkB) kinase (IKK) signalosome. IKK phosphorylates IkB-a at Ser32 and Ser36 in response to a variety of stimuli, resulting in its ubiquitination and subsequent proteasomal degradation. The released NF-kB targets to the nucleus and thereby induces the expression of specific target genes. In addition to nuclear translocation of the NF-kB complex, previous studies have shown that a subunit of NF-kB, RelA/ p65, is post-translationally modified, such as phosphorylation, ubiquitination, or acetylation, and these changes influence its transcriptional activity. In particular, phosphorylation of RelA/ p65 at Ser276, Ser529, or Ser536 could be indispensable for its ability to function as an activator of gene expression. 2 However, recent findings showing a role for Ser529/536 phosphorylation on RelA/p65 activation in response to TNF-a arise conflicting evidences. [3][4][5] In contrast, accumulating evidences have revealed that Ser276 phosphorylation is critical for transactivation of RelA/p65 at least in response to TNF-a. Moreover, Ser276 is the major phosphorylation site of RelA/p65 induced by TNF-a. Phospho-RelA/p65 at Ser276 forms a stable complex with co-activator CBP/p300, a modification apparently required for assembly of a functional enhanceosome on the responsive promoters. 6,7 As nucle...

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Structural Basis of Inhibitor Specificity of the Human Protooncogene Proviral Insertion Site in Moloney Murine Leukemia Virus (PIM-1) Kinase

Cited by 144 publications

References 54 publications

A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases

A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases

Structure and functional characterization of the atypical human kinase haspin

Pim-1 controls NF-κB signalling by stabilizing RelA/p65

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