Protein Phosphorylation And regulation of Cytoskeleton in Nervous system In the nervous system: Neuronal cytoskeletal protein phosphorylation is topographically regulated. Under normal conditions, kinases, phosphatases, cytoskeletal protein substrates, and regulators are synthesized in cell bodies but the phosphorylation of cytoskeletal proteins, particularly medium molecular mass (NF-M) and high molecular mass (NF-H) tail domains, for example, is restricted to the axonal compartment during axon transport. In several neurodegenerative disorders, such as Alzheimer's disease (AD) and Amyotrophic Lateral Sclerosis (ALS), an aberrant phosphorylation of cytoskeletal proteins is found in the cell body. The mechanisms of topographic regulation and deregulation are not well understood and the major focus of this laboratory has been to study of the factors that regulate the phosphorylation of the cytoskeletal proteins. We proposed the following hypotheses to explain the topographic regulation of cytoskeletal proteins; 1) after biosynthesis in the cell bodies, the cytoskeletal proteins are transiently phosphorylated in the N-terminal domains by PKA/PKC. By virtue of conformational changes induced by this phosphorylation, the phosphorylation in the C-terminal domains by proline directed kinases (Cdk5, MAPKs) is inhibited. 2) Exogenous signals, either from the target tissues or from surrounding axon-associated glia, activate the proline-directed kinases, which extensively phosphorylate the proline-directed S/T residues in the axonal compartment. 3) Higher phosphatase activity in the cell body compared to axonal compartment inhibits cytoskeletal protein phosphorylation. Experiments related to the 1st and 2nd hypotheses have been conducted and supporting evidence for these proposals have been published. To address the 3rd hypothesis we are using the squid giant fiber system. We studied the differential phosphatase activities in cell body and axonal compartments that may be responsible for the topographic cytoskeletal protein phosphorylation. We have found that in the squid giant axon system, tyrosine protein phosphatase activity is significantly elevated in the perikarya (cell body) compared to the axonal compartment. Future studies are directed to determine whether tyrosine phosphatases play a role in regulating cell body phosphorylation. In addition, our laboratory has continued to study the regulation and role of Cyclin-dependent kinase 5 (Cdk5) in nervous system development and function. Cdk5 is one of the major kinases that phosphorylate the cytoskeletal proteins in the nervous system and is essential for survival. We, as well as other laboratories, have shown that Cdk5 is a multifunctional protein kinase. The diverse roles of this kinase are based upon its ability to phosphorylate a diverse array of substrates, regulation of other kinases such as MEK1, JNK3, CPRK and GSK3b and ?cross-talk? with other signal transduction cascades such as the Rac signaling pathway. It has been proposed that deregulation of Cdk5 activity in the brain, by abnormal production of p25, a truncated, more active fragment of its regulator p35, can lead to hyperphosphorylated tau, a pathology characteristic of AD. Our study of site specific interactions between Cdk5 and truncated forms of its regulator have revealed a central 125 amino acid fragment termed CIP, has a high affinity for and inhibits the in vitro activity of the Cdk5/p25 complex. We have shown that CIP specifically inhibits Cdk5/p25 activity in transfected non-neuronal and primary neuronal cultures. Additionally, CIP also reduces the hyperphosphorylation of tau. It is important to note that CIP does not affect the activity of Cdc2 kinase nor Cdk5/p35 which is essential for neuronal survival. In addition, we have shown that CIP specifically inhibits Cdk5 hyperactivity in primary neurons. Now the question arises whether CIP can inhibit the hyperphosphorylation of tau and NF proteins in mouse models of AD and ALS, as well as in mice over expressing p25. If CIP does indeed inhibit Cdk5 activity in vivo under these transgenic conditions, then we would predict that the level of tau and NF hyperphosphorylation in the brains of CIP/p25 double transgenics and CIP/AD or CIP/ALS models would be abolished or significantly lower. The outcome of these studies may indicate whether CIP may serve as a therapeutic agent for neuropathologies involving increased Cdk5/p25 activity and the hyperphosphorylation of neuronal cytoskeletal proteins.
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