Signal Transduction in the Brain: Transcript Part 5
Regulation of DARPP-32 phosphorylation, Caseinkinase1 (CK1), Casenkinase 2 (CK2), and CDK5
Regulation of DARPP-32 phosphorylation:
But before going to that big regulatory system, let me show you what happens in terms of the regulation of the DARPP-32 phosphorylation. So here is the DARPP-32 molecule, here is 3 and 34, PKA takes a phosphate from ATP, puts it on a 3 and 34, and the PP2B removes it. And then the state of phosphoric& the molecule in the phosphorylate state then depends on the pressure of the kinase and the phophatase. Now this pathway now is controlled by three other pathways which we have found.
Serine137 is phosphorylated by caseinkinase1 (CK1) which takes phosphate from ATP, puts it on a 137, and once this residue is phosphorylated it causes a change in the properties of the DARPP-32 molecule. Although it still can be phosphorylated by PKA. So the PKA still phosphorylates it but the PP2B can't dephosphorylate it. This means that you are potentiating the dopaminergic pathway. So the signaling pathway, the neurotransmitted pathway, which controls this thing is potentiating the dopamine signaling and antagonizing the glutamate signaling.
The second residue that is phosphorylated is serine102 is phosphorylated by casenkinase 2 which takes phosphate from ATP, puts it on the 102, and this causes a different type of structural change in the DARPP-32 molecule. Now the PP2B can dephosphorylate it, but it is a much better substrate for PKA so it is the same net affect. As soon as it is dephosphorylated it gets rephosphorylated and so&
It turns out that both casenkinase-1 and casenkinase-2, by controlling the state of phosphorylation of the DARPP-32, but through two very different mechanisms, promote dopaminergic signaling and antagonize glutametergic signaling, in the one case by promoting phosphorylation and in the other case by inhibiting dephosphorylation. Both of these effects are substrate-directed. That is to say, these phosphorylations don't affect the inherent activity of PKA and PP2B to act on other substrates, only DARPP-32 is affected.
CDK5 Pathway:
But there is a third pathway, which in some ways is the most interesting to us at the moment. CDK5 takes phospate and puts it on T75, and when it does so, it turns this molecule into an inhibitor of PKA and it inhibits the ability of PKA to phosphorylate any substrate protein. This has very wide ranging effects, which I would now like to summarize for you.
So what we have found is that in whole animals, in whole mice or rats, that under resting conditions T75 is highly phosphorylated and T34 is at a very low level, ____ of phosphorylation. But look what happens when the dopamine signal pathway gets activated. When you activate the dopaminergic pathway, you increase PKA activity. And now we introduce these physiological substrates, all generically lumped into one sum here. So let me go back, under resting conditions, [with] these substrates in a dephosphorylated state, there is low kinase activity and high phosphatase activity. Because the T75 keeps the PK inhibited, you don't get much phosphorylation. And because you don't phosphorylate here, the PP1 is not inhibited and you get high dephosphorylation. So, low phosphorylation and high dephosphorylation.
Now the dopamine system gets activated, you activate PKA. You phosphorylate these substrates. You phosphorylate threonine34, you converted DARPP-32 into PP1 inhibitor, and now you get less dephosphorylation.
And now there is this very elegant positive feedback pathway here, which we have found recently, again, that Angus Naim elucidated. It turns out that when the dopamine activates PKA, it causes an activation of PP2A, and the PP2A then dephosphorylates the T75 which then removes its ability to inhibit PKA, which then allows it to further phosphorylate all these substrates and then to do all these downstream things that it does.
Glutamate control of CDK5 Pathway:
It turns out though that the CDK5 pathway itself is controlled by another neurotransmitter, and that other neurotransmitter is glutamate. So glutamate, by activating CDK5, increases the phosphorylation of T75, which inhibits PKA, which then turns this whole system around so you no longer get dephosphorylation. It just becomes more phosphorylated, and you get more inhibition of PKA. This is a very beautiful positive feedback mechanism that you use by both the dopamine and the glutamate pathways. For example, the glutamate, by activating CDK5, you get more phosphorylation here and by inhibiting PKA, you get less dephosphorylation. So this is a positive feedback pathway for the glutamate system. It is also positive feedback pathway for the dopamine system because when the dopamine activates PKA, you not only get activated PKA but then you shut off this inhibitory system.
This slide summarizes how the two neurotransmitters "fight" each other. The dopamine system is associated with an increase in PKA activity and PP2A activity--a kinase and a phophatase-coupled system. And it is associated with high levels of phosphorylation of many substrates. When the glutamate dominates over the dopamine, then it is a CDK5/PP1 kinase phophatase system that controls things and keeps the system in a low physiological state of phosphorylation.
So this is the still broader picture then. I have now gone into some details about the T34 and the T75 interaction. Casenkinase-1, as I mentioned, inhibits the ability to dephosphorylate PP34 by PP2B. This one potentiates the ability of PKA, the phosphorylate and the T75, and these are fighting each other and these two are modulating those systems.
This again is the master scheme and your pain will soon be over. So here is the system. I am not going to add these. I don't have space or time to put in what we now know about these signaling pathways. I will just tell you one interesting fact&that glutamate activating metabotrophic receptors regulates CDK5 activity which phosphorylates T75 and inhibits PKA. So there are just a few little summarizing comments I want to make about this scheme.
Conclusion:
You will notice that all five of the players that I mentioned at the beginning of my talk, these three classes--glutamate receptor and both classes of dopamine receptor involved in this cross talk, integrating all the information coming in to these nerve cells. The DARPP-32 is a bifunctional compound which can be either a kinase inhibitor or a phophatase inhibitor, depending on the information coming into these cells. There are many intercellular targets which we now have which are obviously very attractive targets for development of therapeutic agents for the treatment of various neurological and psychiatric disorders. And when one considers that this is just one cell type in the brain and that there are lots of other cell types which have comparable signaling pathways. There is a lot of work to be done over the coming years to elucidate how all this information is integrated.
In conclusion, I would like to mention particularly Angus Naim who has been a very close friend and colleague for more than 20 years. He in the process of moving from Rockefeller to Yale University but we continue to work together. And I mentioned most of these folks here in enzymology and molecular biology, cell biology, cytochemistry and electrophysiology. And finally I would like to mention several of the laboratories with whom we have elaborated. Anita Aperia at the Karolinska Institute, Paolo Calbresi in Rome, Richard Huganir and Eric Nestler who were students and post docs in our laboratory, who are now distinguished professors at their own uiversities. Actually Eric has moved to UT Southwestern, and James Surmeier at Northwestern.
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