PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity

Cameron, Angus J.M.; Escribano, Cristina; Saurin, Adrian T.; Kostelecky, Brenda; Parker, Peter J.

doi:10.1038/nsmb.1606

Cited by 122 publications

(133 citation statements)

References 38 publications

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“…These and similar observations (23,(28)(29)(30)(31) have brought up the question about the functional role of ATP binding in pseudokinases. Here we have addressed this question in the JAK2 pseudokinase domain, focusing on its pathogenic mutants.…”

Section: Discussionmentioning

confidence: 92%

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ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

Hammarén

Ungureanu

Grisouard

et al. 2015

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

123

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Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wildtype JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.T he Janus kinases (JAK1-3, TYK2) are a family of nonreceptor tyrosine kinases with essential functions in the regulation of hematopoiesis, the immune system, and cellular metabolism. JAKs interact specifically with various cytokine receptors and couple cytokine binding to cytoplasmic signaling cascades, including the signal transducers and activators of transcription (STAT) pathway. JAKs consist of an N-terminal FERM domain, an SH2-like (Src homology 2) domain, a pseudokinase domain (JAK homology 2, JH2), and the C-terminal tyrosine kinase domain (JH1). JH2 mediates critical regulatory functions in JAKs and primarily serves to inhibit basal JH1 activity. Experimental deletion of JH2 increases JH1 activity in full-length JAK in the absence of stimulation (1-3), and in recombinant systems addition of JH2 suppresses JH1 activity (4-6). JH2 is, however, also required for ligand-induced activation of full-length JAKs in cell (1-3, 6, 7). The regulatory functions of JH2 are corroborated by the multitude of human disease mutations identified in the domain. The most common JAK2 mutation, V617F, leads to cytokine-independent signaling through the exclusively JAK2-dependent homotypic receptors for erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), and thrombopoietin (8). The V617F mutation is found in ∼95% of patients with polycythemia vera (PV) (9...

show abstract

Section: Discussionmentioning

confidence: 92%

“…Protein kinase C, for example, has been shown to be regulated by noncatalytic nucleotide binding (30). Also, in the pseudokinases STRADα (28,36) and integrin-linked kinase (ILK) (23,31), ATP binding is required to enable critical protein-protein interactions.…”

Section: Discussionmentioning

confidence: 99%

ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

Hammarén

Ungureanu

Grisouard

et al. 2015

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

123

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“…Accumulating data indicate that the conformation of the ATP-binding pocket can have a significant impact on catalytic-independent functions of kinases. For example, recent studies with Akt (16) and PKCε (21) suggest that binding to an ATP-competitive inhibitor potentiates membrane recruitment and the induction/stabilization of priming phosphorylation events. In the case of PKCε, priming phosphorylation events for the M486A gatekeeper mutant are compromised upon PMA-induced translocation to the plasma membrane (21).…”

Section: Discussionmentioning

confidence: 99%

“…Besides reductions in activity (k cat ), mutation of the gatekeeper to a small amino acid can have significant effects on substrate binding (K m ). For example, the M486A gatekeeper mutation of PKCε causes a marked decrease in priming site phosphorylation relative to wild type PKCε, an observation that has been attributed to a reduced affinity for ATP (21).…”

Section: Chemical Genetics | Irreversible Inhibitor | Sgk494mentioning

confidence: 99%

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

Garske

Peters

Cortesi

et al. 2011

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

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The conserved nature of the ATP-binding site of the >500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few offtargets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases. chemical genetics | irreversible inhibitor | SgK494

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“…11 critical lysine in the ATP-binding site of these proteins results in increased susceptibility to phosphatases when expressed in cells, rather than defects in the ability of these mutants to autophosphorylate [58]. More recent evidence has demonstrated that the mammalian Target of Rapamycin (mTOR) pathway regulates TM phosphorylation of some, but not all, PKCs [59][60][61].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

Freeley

Kelleher

Long

2011

Cellular Signalling

105

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PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity

Cited by 122 publications

References 38 publications

ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

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