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Cellular Pharmacology of Protein Kinase Mζ (PKMζ) Contrasts with Its in Vitro Profile

IMPLICATIONS FOR PKMζ AS A MEDIATOR OF MEMORY*
  • Alyssa X. Wu-Zhang
    Footnotes
    Affiliations
    Department of Pharmacology, University of California San Diego, La Jolla, California, 92093

    Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California, 92093
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  • Cicely L. Schramm
    Footnotes
    Affiliations
    Department of Pharmacology, University of California San Diego, La Jolla, California, 92093
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  • Sadegh Nabavi
    Affiliations
    Department of Neurosciences, University of California San Diego, La Jolla, California, 92093

    Center for Neural Circuits and Behavior, University of California San Diego, La Jolla, California 92093
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  • Roberto Malinow
    Correspondence
    To whom correspondence may be addressed: 9500 Gilman Dr., La Jolla, CA 92093-0634. Tel.: 858-246-0278;
    Affiliations
    Department of Neurosciences, University of California San Diego, La Jolla, California, 92093

    Center for Neural Circuits and Behavior, University of California San Diego, La Jolla, California 92093
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  • Alexandra C. Newton
    Correspondence
    To whom correspondence may be addressed: 9500 Gilman Dr., La Jolla, CA 92093-0721. Tel.: 858-534-4527; Fax: 858-822-5888;
    Affiliations
    Department of Pharmacology, University of California San Diego, La Jolla, California, 92093
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    2 Supported in part by the University of California San Diego (UCSD) Graduate Training Program in Cellular and Molecular Pharmacology through Institutional Training Grant T32 GM007752 from the NIGMS, National Institutes of Health, and in part by a National Science Foundation (NSF) GK-12 STEM fellowship in education at UCSD, PI Maarten Chrispeels, NSF 0742551.
    3 Supported by the National Institutes of Health/NIDDK Hemoglobin and Blood Protein Chemistry Training Grant 5T32-DK007233.
    * This work was supported, in whole or in part, by National Institutes of Health Grants GM-43154 (to A. C. N.) and MH-049159 (to R. M.). This work was also supported by Cure Alzheimer's Foundation (to R. M.).
Open AccessPublished:February 29, 2012DOI:https://doi.org/10.1074/jbc.M112.357244
      A number of recent studies have used pharmacological inhibitors to establish a role for protein kinase Mζ (PKMζ) in synaptic plasticity and memory. These studies use zeta inhibitory peptide (ZIP) and chelerythrine as inhibitors of PKMζ to block long term potentiation and memory; staurosporine is used as a negative control to show that a nonspecific kinase inhibitor does not block long term potentiation and memory. Here, we show that neither ZIP nor chelerythrine inhibits PKMζ in cultured cells or brain slices. In contrast, staurosporine does block PKMζ activity in cells and brain slices by inhibiting its upstream phosphoinositide-dependent kinase-1. These studies demonstrate that the effectiveness of drugs against purified PKMζ may not be indicative of their specificity in the more complex environment of the cell and suggest that PKMζ is unlikely to be the mediator of synaptic plasticity or memory.

      Introduction

      Synaptic plasticity is generally thought to be the cellular correlate of memory (
      • Squire L.R.
      • Kandel E.R.
      ). In particular, long term potentiation (LTP)
      The abbreviations used are: LTP
      long term potentiation
      bisIV
      bisindolylmaleimide IV
      CKAR
      C kinase activity reporter
      DMSO
      dimethyl sulfoxide
      PDK1
      phosphoinositide-dependent kinase-1
      PKCζ
      protein kinase Cζ
      PKMζ
      catalytic domain of PKCζ
      ZIP
      zeta inhibitory peptide.
      of synaptic transmission, which is triggered by a brief high frequency activation of synapses, is commonly thought to account for the maintenance of memory. Research directed toward understanding the molecular basis of LTP and memory has firmly established the role of such molecules as synaptic NMDA receptors and such processes as a rise in intracellular calcium in triggering synaptic plasticity (
      • Squire L.R.
      • Kandel E.R.
      ). Subsequent biochemical events, especially those that maintain LTP and memory, however, have not been well established. One proposal has been that the transient rise in intracellular calcium leads to production of a brain-specific alternative transcript of protein kinase Cζ (PKCζ) that encodes only the catalytic domain (PKMζ) (
      • Hernandez A.I.
      • Blace N.
      • Crary J.F.
      • Serrano P.A.
      • Leitges M.
      • Libien J.M.
      • Weinstein G.
      • Tcherapanov A.
      • Sacktor T.C.
      Protein kinase Mζ synthesis from a brain mRNA encoding an independent protein kinase Cζ catalytic domain: implications for the molecular mechanism of memory.
      ). This persistently active enzyme could phosphorylate downstream activators and maintain plasticity and memory beyond the initial triggering events (
      • Ling D.S.
      • Benardo L.S.
      • Serrano P.A.
      • Blace N.
      • Kelly M.T.
      • Crary J.F.
      • Sacktor T.C.
      Protein kinase Mζ is necessary and sufficient for LTP maintenance.
      ,
      • Pastalkova E.
      • Serrano P.
      • Pinkhasova D.
      • Wallace E.
      • Fenton A.A.
      • Sacktor T.C.
      Storage of spatial information by the maintenance mechanism of LTP.
      ,
      • Shema R.
      • Sacktor T.C.
      • Dudai Y.
      Rapid erasure of long term memory associations in the cortex by an inhibitor of PKMζ.
      ,
      • Serrano P.
      • Friedman E.L.
      • Kenney J.
      • Taubenfeld S.M.
      • Zimmerman J.M.
      • Hanna J.
      • Alberini C.
      • Kelley A.E.
      • Maren S.
      • Rudy J.W.
      • Yin J.C.
      • Sacktor T.C.
      • Fenton A.A.
      PKMζ maintains spatial, instrumental, and classically conditioned long term memories.
      ,
      • Migues P.V.
      • Hardt O.
      • Wu D.C.
      • Gamache K.
      • Sacktor T.C.
      • Wang Y.T.
      • Nader K.
      PKMζ maintains memories by regulating GluR2-dependent AMPA receptor trafficking.
      ).
      Evidence that PKMζ is required for LTP and for the maintenance of several forms of memory has depended almost exclusively on the use of pharmacological approaches (
      • Ling D.S.
      • Benardo L.S.
      • Serrano P.A.
      • Blace N.
      • Kelly M.T.
      • Crary J.F.
      • Sacktor T.C.
      Protein kinase Mζ is necessary and sufficient for LTP maintenance.
      ,
      • Pastalkova E.
      • Serrano P.
      • Pinkhasova D.
      • Wallace E.
      • Fenton A.A.
      • Sacktor T.C.
      Storage of spatial information by the maintenance mechanism of LTP.
      ,
      • Shema R.
      • Sacktor T.C.
      • Dudai Y.
      Rapid erasure of long term memory associations in the cortex by an inhibitor of PKMζ.
      ,
      • Serrano P.
      • Friedman E.L.
      • Kenney J.
      • Taubenfeld S.M.
      • Zimmerman J.M.
      • Hanna J.
      • Alberini C.
      • Kelley A.E.
      • Maren S.
      • Rudy J.W.
      • Yin J.C.
      • Sacktor T.C.
      • Fenton A.A.
      PKMζ maintains spatial, instrumental, and classically conditioned long term memories.
      ,
      • Migues P.V.
      • Hardt O.
      • Wu D.C.
      • Gamache K.
      • Sacktor T.C.
      • Wang Y.T.
      • Nader K.
      PKMζ maintains memories by regulating GluR2-dependent AMPA receptor trafficking.
      ). These studies have relied on the use of zeta inhibitory peptide (ZIP), a myristoylated putative PKCζ-inhibiting peptide derived from the autoinhibitory pseudosubstrate peptide sequence within PKCζ, chelerythrine, an apoptosis-inducing compound that is marketed, and extensively used, as a PKC inhibitor (
      • Herbert J.M.
      • Augereau J.M.
      • Gleye J.
      • Maffrand J.P.
      Chelerythrine is a potent and specific inhibitor of protein kinase C.
      ,
      • Yamamoto S.
      • Seta K.
      • Morisco C.
      • Vatner S.F.
      • Sadoshima J.
      Chelerythrine rapidly induces apoptosis through generation of reactive oxygen species in cardiac myocytes.
      ), and staurosporine, a general protein kinase inhibitor (
      • Karaman M.W.
      • Herrgard S.
      • Treiber D.K.
      • Gallant P.
      • Atteridge C.E.
      • Campbell B.T.
      • Chan K.W.
      • Ciceri P.
      • Davis M.I.
      • Edeen P.T.
      • Faraoni R.
      • Floyd M.
      • Hunt J.P.
      • Lockhart D.J.
      • Milanov Z.V.
      • Morrison M.J.
      • Pallares G.
      • Patel H.K.
      • Pritchard S.
      • Wodicka L.M.
      • Zarrinkar P.P.
      A quantitative analysis of kinase inhibitor selectivity.
      ). ZIP and chelerythrine have been found to block LTP and memory, theoretically by inhibiting PKMζ, whereas staurosporine, which does not inhibit purified PKMζ in vitro, fails to do so. Beyond their in vitro testing against pure protein, however, the effectiveness of ZIP or chelerythrine and the ineffectiveness of staurosporine in inhibiting PKMζ within the complex milieu of mammalian cells and tissues have never been established. Indeed, studies show that the cellular effects of chelerythrine are not mediated by PKC (
      • Yu R.
      • Mandlekar S.
      • Tan T.H.
      • Kong A.N.
      Activation of p38 and c-Jun N-terminal kinase pathways and induction of apoptosis by chelerythrine do not require inhibition of protein kinase C.
      ), nor does the compound inhibit PKC isoforms (or any other kinase in a screen of 34 structurally diverse kinases) in vitro (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      Specificity and mechanism of action of some commonly used protein kinase inhibitors.
      ,
      • Thompson L.J.
      • Fields A.P.
      βII protein kinase C is required for the G2/M phase transition of cell cycle.
      ) or in cells (
      • Gould C.M.
      • Antal C.E.
      • Reyes G.
      • Kunkel M.T.
      • Adams R.A.
      • Ziyar A.
      • Riveros T.
      • Newton A.C.
      Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form.
      ). Furthermore, the effectiveness of pseudosubstrate peptides in binding and inhibiting an enzyme depends largely on their intramolecular nature, an advantage not possessed by ZIP. And lastly, staurosporine, though it may not inhibit PKMζ directly in vitro (
      • Ling D.S.
      • Benardo L.S.
      • Serrano P.A.
      • Blace N.
      • Kelly M.T.
      • Crary J.F.
      • Sacktor T.C.
      Protein kinase Mζ is necessary and sufficient for LTP maintenance.
      ,
      • Seynaeve C.M.
      • Kazanietz M.G.
      • Blumberg P.M.
      • Sausville E.A.
      • Worland P.J.
      Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue.
      ), does inhibit phosphoinositide-dependent kinase-1 (PDK1) (
      • Karaman M.W.
      • Herrgard S.
      • Treiber D.K.
      • Gallant P.
      • Atteridge C.E.
      • Campbell B.T.
      • Chan K.W.
      • Ciceri P.
      • Davis M.I.
      • Edeen P.T.
      • Faraoni R.
      • Floyd M.
      • Hunt J.P.
      • Lockhart D.J.
      • Milanov Z.V.
      • Morrison M.J.
      • Pallares G.
      • Patel H.K.
      • Pritchard S.
      • Wodicka L.M.
      • Zarrinkar P.P.
      A quantitative analysis of kinase inhibitor selectivity.
      ,
      • Komander D.
      • Kular G.S.
      • Bain J.
      • Elliott M.
      • Alessi D.R.
      • Van Aalten D.M.
      Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition.
      ), the upstream kinase whose constitutive phosphorylation of all PKC isoforms is required for their kinase activity (
      • Dutil E.M.
      • Toker A.
      • Newton A.C.
      Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase-1 (PDK-1).
      ,
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1.
      ,
      • Newton A.C.
      Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm.
      ). The discrepancies between the potential effectiveness of these compounds in inhibiting PKMζ activity in cells and their reported effects on learning and memory suggest that PKMζ is not mediating the effects of these drugs in cells.
      Here, we examine whether the inhibitors used to implicate PKMζ in learning and memory block PKMζ activity in the context of heterologous cells and brain slices. We demonstrate that those inhibitors that have been reported to impact learning and memory, ZIP and chelerythrine, do not inhibit PKMζ in cells, whereas an inhibitor reported not to impact learning and memory, staurosporine, does inhibit PKMζ in cells. These data indicate that PKMζ is not the cellular target of ZIP or chelerythrine, that PKMζ is a cellular target of staurosporine, and that PKMζ likely does not mediate learning or memory.

      DISCUSSION

      Identification of the molecules responsible for the long lasting maintenance of memory continues to be an important biological question. A number of recent studies have used pharmacological approaches to implicate the action of PKMζ as a potential mechanism (
      • Ling D.S.
      • Benardo L.S.
      • Serrano P.A.
      • Blace N.
      • Kelly M.T.
      • Crary J.F.
      • Sacktor T.C.
      Protein kinase Mζ is necessary and sufficient for LTP maintenance.
      ,
      • Pastalkova E.
      • Serrano P.
      • Pinkhasova D.
      • Wallace E.
      • Fenton A.A.
      • Sacktor T.C.
      Storage of spatial information by the maintenance mechanism of LTP.
      ,
      • Shema R.
      • Sacktor T.C.
      • Dudai Y.
      Rapid erasure of long term memory associations in the cortex by an inhibitor of PKMζ.
      ,
      • Serrano P.
      • Friedman E.L.
      • Kenney J.
      • Taubenfeld S.M.
      • Zimmerman J.M.
      • Hanna J.
      • Alberini C.
      • Kelley A.E.
      • Maren S.
      • Rudy J.W.
      • Yin J.C.
      • Sacktor T.C.
      • Fenton A.A.
      PKMζ maintains spatial, instrumental, and classically conditioned long term memories.
      ,
      • Migues P.V.
      • Hardt O.
      • Wu D.C.
      • Gamache K.
      • Sacktor T.C.
      • Wang Y.T.
      • Nader K.
      PKMζ maintains memories by regulating GluR2-dependent AMPA receptor trafficking.
      ). To our knowledge, the effectiveness of ZIP, chelerythrine, or staurosporine in inhibiting PKMζ within the complex milieu of mammalian cells and tissues has not been previously demonstrated. Here, we find that ZIP and chelerythrine fail to inhibit PKMζ activity in heterologous cells or brain slices. Based on the oriented peptide library work of Cantley and co-workers (
      • Nishikawa K.
      • Toker A.
      • Johannes F.J.
      • Songyang Z.
      • Cantley L.C.
      Determination of the specific substrate sequence motifs of protein kinase C isozymes.
      ), the sequences of ZIP (SIYRRGARRWRKL), which is identical to that of the PKCζ pseudosubstrate, and scrambled ZIP (RLYRKRIWRSAGR), when substituted with Ser at the phosphoacceptor position (underlined), are predicted to be equally good substrates for PKCζ, which may explain the partial inhibition of PKCζ in vitro even by scrambled ZIP (Fig. 1C), a negative control peptide. Furthermore, the pseudosubstrate sequence of PKCζ, when substituted with Ser at the phosphoacceptor site, is actually phosphorylated with an order of magnitude lower Km and an order of magnitude higher Vmax/Km by a different PKC isozyme, PKCδ (
      • Nishikawa K.
      • Toker A.
      • Johannes F.J.
      • Songyang Z.
      • Cantley L.C.
      Determination of the specific substrate sequence motifs of protein kinase C isozymes.
      ). Thus, the lack of a defined substrate consensus sequence for PKC isozymes, including PKCζ, precludes the use of pseudosubstrate peptides as specific pharmacological tools. Because ZIP clearly affects LTP, we conclude that this basic peptide likely disrupts a macromolecular interaction in cells via a target unrelated to PKMζ. Finally, we find that staurosporine actually does inhibit PKMζ in cells and brain slices by inhibiting its constitutive phosphorylation by the upstream kinase PDK1. This effect of staurosporine would be overlooked in in vitro assays, from which PDK1 would be absent, because the purified PKMζ used would already be phosphorylated at the PDK1 site and thus catalytically competent.
      Measurement of the phosphorylation of multiple distinct endogenous PKMζ substrates in cell lines and hippocampal brain slices as well as an overexpressed PKMζ substrate in the form of CKAR all show that, in contrast to their effects in vitro, ZIP and chelerythrine do not and staurosporine actually does inhibit PKMζ in the complex milieu of mammalian cells and tissues. The inconsistencies between the effectiveness of ZIP, chelerythrine, and staurosporine in inhibiting PKMζ activity in cells and tissues and their reported effects on learning and memory provide a double dissociation between PKMζ activity and synaptic plasticity. We note that genetic studies in which PKMζ has been overexpressed have been used to support a role for PKMζ in memory (
      • Ling D.S.
      • Benardo L.S.
      • Serrano P.A.
      • Blace N.
      • Kelly M.T.
      • Crary J.F.
      • Sacktor T.C.
      Protein kinase Mζ is necessary and sufficient for LTP maintenance.
      ,
      • Shema R.
      • Haramati S.
      • Ron S.
      • Hazvi S.
      • Chen A.
      • Sacktor T.C.
      • Dudai Y.
      Enhancement of consolidated long term memory by overexpression of protein kinase Mζ in the neocortex.
      ). Given that overexpressed kinases can mislocalize and increase the phosphorylation of both specific and nonspecific substrates in cells and given that these reports have not included specificity controls, their results are inconclusive. Because the cellular pharmacology of PKMζ contrasts with its in vitro profile, we conclude that PKMζ cannot be implicated as the molecular substrate of long term plasticity or memory based on the prevalent studies using the pharmacological tools of ZIP, chelerythrine, and staurosporine.

      Acknowledgments

      We thank Matt Niederst and Maya Kunkel for performing the apoptosis assay in Fig. 1D.

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