PROTEIN SERINE THREONINE PHOSPHATASE-1 AND 2A ASSOCIATE WITH AND DEPHOSPHORYLATE NEUROFILAMENTS/

The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon dia meter. Immunocytochemical analyses of spinal cord revealed axonal loca lization of all protein phosphatase subunits. To determine whether pro tein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel fil tration. approximate to 15% of the total phosphorylase a phosphatase a ctivity was present in the neurofilament fraction. The catalytic subun its of PPI and PP2A, as well as the A and B alpha regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting , whereas PP2B and PP2C were found exclusively in the low molecular we ight soluble fractions. PP1 and PP2A subunits could be partially disso ciated from neurofilaments by high salt but not by phosphatase inhibit ors, indicating that the interaction does not involve the catalytic si te. In both neurofilament and soluble fractions, 75% of the phosphatas e activity towards exogenous phosphorylase a could be attributed to PP 2A, and the remainder to PPI as shown with specific inhibitors. Neurof ilament proteins were phosphorylated in vitro by associated protein ki nases which appeared to include protein kinase A, calcium/calmodulin-d ependent protein kinase, and heparin-sensitive and -insensitive cofact or-independent kinases. Dephosphorylation of phosphorylated neurofilam ent subunits was mainly (60%) catalyzed by associated PP2A, with PP1 c ontributing minor activity (10-20%). These studies suggest that neurof ilament-associated PPI and PP2A play an important role in the regulati on of neurofilament phosphorylation. (C) 1997 Elsevier Science B.V.