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

Nature Structural & Molecular Biology volume 16, pages 624–630 (2009)Cite this article

Abstract

The protein kinase C (PKC) Ser/Thr kinases account for ∼2% of the human kinome and regulate diverse cellular behaviors. PKC catalytic activity requires priming phosphorylations at three conserved sites within the kinase domain. Here we demonstrate that priming of PKC is dependent on the conformation of the nucleotide binding pocket but not on its intrinsic kinase activity. Inactive ATP binding site mutants are unprimed, but they become phosphorylated upon occupancy of the ATP binding pocket with inhibitors of PKC. We have exploited this property to screen for PKC inhibitors in vivo. Further, we generated a distinct class of kinase-inactive mutants that maintain the integrity of the ATP binding pocket; such mutants are constitutively primed and functionally distinct from ATP binding site mutants. These data demonstrate that autophosphorylation is not required for PKC priming and show how ATP pocket occupation can enable a kinase to mature as well as function.

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Figure 1: ATP-competitive inhibitors induce priming of kinase-inactive PKCε K437M.
Figure 2: Characterization of inhibitor-induced PKCε K437M phosphorylation.
Figure 3: Phosphorylation of PKCε is directly induced by occupation of the nucleotide binding pocket.
Figure 4: Inhibitor-induced priming of PKCε mutants is protein autonomous.
Figure 5: Redesigning kinase-inactive PKC to retain wild-type (WT) ATP binding rescues priming phosphorylations.

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Acknowledgements

We thank R. Messing for the characterization of PKCε M486A chemical genetic mutants, M. Howell for assistance with the Calbiochem Inhibitor library screen, A. Hibbert for statistical advice and R. Mitter for help with data presentation. We also acknowledge N. MacDonald, M. De Rycker, W. Hanage and A. Jevons for critical reading of the manuscript. C.E. gratefully acknowledges the support of Jose Luis Rodríguez-Fernández (Madrid).

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Authors and Affiliations

  1. Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London, UK

    Angus J M Cameron, Cristina Escribano, Adrian T Saurin & Peter J Parker

  2. Centro de Investigaciones Biológicas, Madrid, Spain

    Cristina Escribano

  3. Structural Biology Laboratory, London Research Institute, Cancer Research UK, London, UK

    Brenda Kostelecky

  4. Division of Cancer Studies, King's College London, London, UK

    Peter J Parker

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  1. Angus J M Cameron
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Contributions

A.J.M.C. carried out all the experiments in this study; A.J.M.C. jointly conceived the project (with P.J.P.) and wrote the manuscript with help from P.J.P.; C.E. helped with phosphomimetic mutants of PKC.; A.T.S. helped with PKCε constructs and tetracycline-inducible cell lines; B.K. contributed structural analysis of kinase domains.

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Correspondence to Peter J Parker.

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Cameron, A., Escribano, C., Saurin, A. et al. PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity. Nat Struct Mol Biol 16, 624–630 (2009). https://doi.org/10.1038/nsmb.1606

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