Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry

In a recent study:

Wu-Zhang, Schramm, Nabavi, Malinow, Newton (2012). Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry.

At first I skimmed through and saw they used FRET and thought it would be easy one to argue against. Then I read it. ZIP doesn’t affect the phosphorylation rate of PKMzeta (on any substrate empirically shown to be phosphorylated by PKMz). They showed this effect using simple immunoblotting methods, not just FRET. I can’t believe this hasn’t been done already. Do you have any arguments against the findings?

Summary of Experiment Here

Dr. Alexandra Newton (the article author) who’s research focuses on the functional mechanisms of phospholipase and kinase (PLC & PI3K) signaling pathways; and Dr. Todd Sacktor, who’s a long-standing pioneer of research on protein kinase C (PKC) isozymes in learning and memory.

**UPDATE 2**
This publication may be considered a direct response by Yao and Sacktor to the Wu-Zhang paper mentioned above

Matching biochemical and functional efficacies confirm ZIP as a potent competitive inhibitor of PKMz in neurons by Yudong Yao, Charles Shao, Desingarao Jothianandan, Andrew Tcherepanov, Harel Shouval, Todd Charlton Sacktor*

7 thoughts on “Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry

  1. In J Neuroscience 25:1979-1984 (2005) and Nature Neuroscience 5:295-296 (2002), ZIP and chelerythrine were shown to block the effect of injected PKMζ in neurons. So it has previously been demonstrated that they are effective inhibitors of PKMζ, both in vitro and in cells.

    There are several reasons for the negative results of this paper. All the experiments were with overexpression of PKMζ. In neurons, this typically increases PKMζ amount by >10-fold over normal amounts. But they use the same concentrations of inhibitors that were used when injecting very low amounts (1-20 nanomolar) of the kinase into neurons (in the references cited above). When an enzyme is overexpressed so that it begins to saturate substrate, a higher dose of a competitive inhibitor is required. In fact, ZIP inhibits overexpressed PKMζ in neurons at a higher dose (Hippocampus. 2011 Dec 23).

    Other concerns are the RFP-fusion to the C-terminal of PKMζ, which will mislocalize the protein, and the unidentified or overexpressed nature of the substrates. ZIP and chelerythrine are both competitive inhibitors, so abundant or high affinity substrates will again require higher doses.

    The proper experiment is to identify and study the effect of the inhibitors on the physiologically relevant substrates of PKMζ that mediate its ability to enhance synaptic transmission.

  2. Thanks for the reply Todd. I was also concerned about the RFP-fusion to the C-terminus, but it appears to have no trouble with substrate phosphorylation. Looking at the immunoblotting data the effect of over-expressed PKMz was increased 5-fold with no effect of 1 uM ZIP. What do you think would be an appropriate amount of ZIP to convince you this is a real ‘non’-effect?

  3. Bradley, The question is how much overexpression was there in the cell, relative to the amount of kinase that saturates the substrate. Those data were not provided. Imagine there is 10-fold excess kinase. Then inhibiting 90% of the kinase will still have no effect on substrate phosphorylation.

    Modeling indicates that when a kinase saturates a substrate, the IC50 of the drug becomes linear with the enzyme concentration. As mentioned above, published data have already shown that ZIP blocks PKMzeta in cells. 5 micromolar was the appropriate dose to start with based on these published experiments, not 1 micromolar. Then they should have measured the amount of overexpression. In our hands, using Sindbis virus in neurons, which they used, the increase is 10-20 times endogenous levels. So to inhibit 10-20 times saturated levels (assuming the endogenous amount is close to saturation) means 50-100 micromolar. Not 1 micromolar.

  4. Those are actually great points Todd. I’m working with ZIP right now so, I’m very familiar with your work. I’ve invited the authors to comment on your posts. We will see if they have anything to say on the matter.

  5. Hi Bradley – thanks for your interest in our study. We were also surprised that the effect of these reagents on the kinase activity of PKM zeta in cells had not been measured previously (the studies referenced did not measure cellular kinase activity). There are two issues brought up above. First, whether we overexpressed PKM zeta to such a high level that the pseudosubstrate peptide was ineffective. If you look at the data in our paper, you will note that ZIP had no effect on the phosphorylation of a PKM zeta target (MARK2) catalyzed by endogenous kinase. Also note we used 10 µM ZIP in the study (not just 1 µM). Note also that ZIP did enter cells and actually increased phosphorylation of MARK2. Second, there was a question about the tag being on the C-terminus. As you pointed out, this construct is active. When proteins are overexpressed to this level (10x over endogenous), they mislocalize and the problem is that they start recognizing new substrates rather than not recognizing physiological substrates.
    More relevant to your discussions are that 1: staurosporine potently inhibits PKM zeta in cells by inhibiting the upstream activating kinase PDK-1 and 2: chelerythrine is not a protein kinase inhibitor. Since staurosporine did not block established LTP or memory, this argues that PKM zeta is nor responsible. The use of chelerythrine says nothing about the role of any protein kinase. There were also data presented at SFN from Rick Huganir’s lab indicating that PKC zeta knockout mice have normal LTP and ZIP has the same effect on the knockout as in the wild type. All together, these data indicate that PKM zeta is not the target of ZIP. It will be interesting to find the real target.

  6. Thank you very much for the reply Alexandra. I do remember the Huganir data at SfN. At this point, I’m wondering how difficult it would be to build a designer PKMz inhibitor where there’s no question that it selectively blocks PKMz phosphorylation.

  7. Bradley and Alexandra, I’ll just make a few points.
    1) ZIP was actually used on cells by Suzuki et al., in Current Biology, 14, 1425–1435 (2004) to document that MARK2/PAR1b is a substrate of aPKC (Supp Fig.1C). As expected, they used higher concentrations.
    2) Chelerythrine is clearly an inhibitor of the catalytic domain of PKC; the in vitro studies have been reproduced many times, including Nature Neuroscience 5:295-296 (2002).
    3) We have found that staurosporine at the doses we used in hippocampal slices does not alter the state of phosphorylation of the PDK1 site in endogenous PKMζ in slices. Therefore, it is an appropriate control drug.
    4) A conditonal KO of PKC/PKMζ performed in adult animals has no late-phase LTP. Whether there is compensation in the complete or CaMKII promoter-driven KO from another PKC to explain Huganir’s remains to be determined.

    Points 2-4 will be presented at SFN; I hope to see you both there.

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