PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

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Our progress to understand the structure and function of neurofilaments, (NFs), their phosphorylation and to identify the specific kinases and phosphatases involved is as follows: (1) We have shown that second messenger~dependent protein kinases (PK) phosphorylate the head domains and second messenger~independent PKs, casein kinase I and II and microtubule~associated PK~like activities associated with neurofilament preparation phosphorylate serine residues in the C~terminal tail domain of neurofilament proteins (NFPs) in vitro but not the multiple repeat lys~ser~pro (KSP) motifs in middle (NF~M) and high (NF~H) NFPs. (2) The analysis of the phosphorylation state of KSP repeats by means of a combination of chemical and enzymatic digestion, reverse phase high~pressure chromatography, Edman microse~quencing and electrospray mass spectrometry showed that most of the KSP motifs in NF~H are phos~phorylated in vivo; and that the domain containing uninterrupted KSP repeats is highly resistant to proteolysis and can be proteolysed after dephosphorylation. (3) We have identified and isolated from rat spinal cord a protein kinase that phosphorylates a specific KSP sequence (KSPXK) in NF~M and NF~H. Characterization of this enzyme revealed a close relationship to the cell~cycle dependent kinases (CDK), most closely to CDK5. Purification of this CDK5~like kinase from rat spinal cord has shown that it is strongly associated with a protein of 62 kDa (p62). Separation of this protein from the kinase resulted in a considerable decrease in this kinase activity which could be restored by adding back the purified p62. The complete amino acid sequence of p62 was deduced from a number of cDNA clones from rat brain libraries. No similarity exists with any known protein in the current protein sequence data banks. Ribo~probe in situ hybridization experiments of mouse embryos and adults have demonstrated that p62 transcript expression begins early in development and is restricted to the nervous system, exclusively expressed in neurons and absent from surrounding glia. These studies suggest that CDK~like kinases and related regulators (p62) are involved in phosphorylation of KSP sites in NF~M and NF~H, and may be involved in neuronal growth and differentiation as well as in the stability of axonal structures. (4) We have also demonstrated that the phosphorylation of NF~H by CDK5~like kinase is dephosphorylated by protein phosphatase 2A; and identified such a phosphatase in the rat spinal cord.