Myelin is a multilamellar membrane that surrounds axons in both the central and peripheral nervous system P0, a member of the immunoglobulin family of adhesion molecules, is the most abundant protein in peripheral myelin and is thought to mediate adhesion between the multiple layers of myelin as they wrap around the axon. Consistent with an important role for P0 in myelination, mutations in both the extracellular and intracellular domains cause the human peripheral neuropathy Charcot-Marie-Tooth disease type 1B. Our long-range goals are to couple basic and clinical research to discover the molecular basis for P0 function in human myelination. We have recently discovered that point mutations in a Protein Kinase C (PKC) target motif - RSTK - in the cytoplasmic domain of P0 abolish its adhesive function, as does inhibition of PKC . We have also identified a CMT1B patient with a mutation in the RSTK motif (R to S), further indicating the importance of PKC-mediated phosphorylation in myelination. The goals of this proposal are to determine the role of PKC-mediated phosphorylation in adhesion and myelination. 1) We have found that RACK1, the Receptor for Activated C Kinase is a component of the complex of proteins associated with the cytoplasmic domain of P0. We will determine if RACK1 or other adapters are needed to target PKC to the cytoplasmic domain of P0. We will further characterize the domains through which partners interact using deletion constructs in conjunction with the two-hybrid system and/or an in vitro binding assay 2. By analogy with other adhesion molecules, we hypothesize that phosphorylation creates a binding site for adaptors or effectors essential for the interaction of P0 with downstream targets. We have identified one protein whose interaction with P0 depends on phosphorylation of serine in the RSTK motif using the yeast two-hybrid system. We will characterize the binding of this component and downstream targets. 3) We will evaluate the role of each of the components identified in aims 1 and 2 using an in vitro myelination culture system by introducing dominant-negative and constitutively active constructs, as well as constructs coding for peptide competitors for critical protein-protein interactions, into Schwann cells prior to co-culture with neurons.
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