Comments for Musings on Life, Technology, and Neuroscience http://www.bradleymonk.com/wp by Bradley Monk Mon, 23 Apr 2012 20:36:39 +0000 hourly 1 http://wordpress.org/?v=3.3.2 Comment on Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry by Todd Sacktor http://www.bradleymonk.com/wp/?p=43&cpage=1#comment-1343 Todd Sacktor Mon, 23 Apr 2012 20:36:39 +0000 http://www.bradleymonk.com/wp/?p=43#comment-1343 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. 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.

]]> Comment on Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry by Bradley http://www.bradleymonk.com/wp/?p=43&cpage=1#comment-1342 Bradley Mon, 23 Apr 2012 19:28:36 +0000 http://www.bradleymonk.com/wp/?p=43#comment-1342 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. 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.

]]> Comment on Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry by Alexandra Newton http://www.bradleymonk.com/wp/?p=43&cpage=1#comment-1340 Alexandra Newton Sun, 22 Apr 2012 23:20:21 +0000 http://www.bradleymonk.com/wp/?p=43#comment-1340 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. 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.

]]> Comment on Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry by Bradley http://www.bradleymonk.com/wp/?p=43&cpage=1#comment-1339 Bradley Sun, 22 Apr 2012 06:12:29 +0000 http://www.bradleymonk.com/wp/?p=43#comment-1339 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. 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.

]]> Comment on Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry by Todd Sacktor http://www.bradleymonk.com/wp/?p=43&cpage=1#comment-1338 Todd Sacktor Sun, 22 Apr 2012 04:58:35 +0000 http://www.bradleymonk.com/wp/?p=43#comment-1338 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. 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.

]]> Comment on Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry by Bradley http://www.bradleymonk.com/wp/?p=43&cpage=1#comment-1337 Bradley Sat, 21 Apr 2012 21:52:07 +0000 http://www.bradleymonk.com/wp/?p=43#comment-1337 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? 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?

]]> Comment on Comments on Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry by Todd Sacktor http://www.bradleymonk.com/wp/?p=43&cpage=1#comment-1336 Todd Sacktor Sat, 21 Apr 2012 04:00:16 +0000 http://www.bradleymonk.com/wp/?p=43#comment-1336 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. 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.

]]> Comment on Corporate Greed. What the shit? by Ryan http://www.bradleymonk.com/wp/?p=34&cpage=1#comment-1333 Ryan Tue, 03 Apr 2012 18:58:41 +0000 http://www.bradleymonk.com/wp/?p=34#comment-1333 Preach on brotha! Love it. Preach on brotha! Love it.

]]> Comment on Why Kill, Why not capture Bin Laden? by Ben B http://www.bradleymonk.com/wp/?p=22&cpage=1#comment-676 Ben B Tue, 03 May 2011 23:35:27 +0000 http://www.bradleymonk.com/wp/?p=22#comment-676 Id say those are 2 fair questions. Shouldn't we (being "democratic") have captured him and be conducting a fair trial? As for the burial at sea, I believe the official statement was to honor his culture's muslim burial traditions. Anyway, the means in which the operation reportedly happened puts the government in a position to be free of providing the public with ANY EVIDENCE that bin laden is actually dead. And I think that is the point. The reality of the whole matter is that it makes no significant difference if bin laden is dead or not. We aren't addressing the problem, we are just seeking revenge on people that do us harm. To murder a figurehead doesn't solve the problem, in fact, it typically just creates more hostility towards the group that murdered him. We should be asking why this group of people called "al qaeda" is hostile towards us in the first place. And by that I dont mean to just take the mainstream medias word for it that "they hate our freedoms", that's absurd. Actually, we should be asking why a large portion of the world's population doesn't like the USA's government policies. Perhaps we would then get some perspective on these issues. Also, if we are truly a democracy, why don't we ever invite the opposing leaders from other countries and groups to have a discussion with our president on live television so all US citizens could develop their personal opinion based on both sides of the story instead of just being given one side of the story. Id say those are 2 fair questions. Shouldn’t we (being “democratic”) have captured him and be conducting a fair trial? As for the burial at sea, I believe the official statement was to honor his culture’s muslim burial traditions. Anyway, the means in which the operation reportedly happened puts the government in a position to be free of providing the public with ANY EVIDENCE that bin laden is actually dead. And I think that is the point.

The reality of the whole matter is that it makes no significant difference if bin laden is dead or not. We aren’t addressing the problem, we are just seeking revenge on people that do us harm. To murder a figurehead doesn’t solve the problem, in fact, it typically just creates more hostility towards the group that murdered him. We should be asking why this group of people called “al qaeda” is hostile towards us in the first place. And by that I dont mean to just take the mainstream medias word for it that “they hate our freedoms”, that’s absurd. Actually, we should be asking why a large portion of the world’s population doesn’t like the USA’s government policies. Perhaps we would then get some perspective on these issues. Also, if we are truly a democracy, why don’t we ever invite the opposing leaders from other countries and groups to have a discussion with our president on live television so all US citizens could develop their personal opinion based on both sides of the story instead of just being given one side of the story.

]]> Comment on Liberal Education in the USA by Bradley http://www.bradleymonk.com/wp/?p=5&cpage=1#comment-14 Bradley Sat, 04 Dec 2010 06:18:44 +0000 http://www.bradleymonk.com/wp/?p=5#comment-14 checking checking

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