Specific phosphorylation of Torpedo 43K rapsyn by endogenous kinase(s) with thiamine triphosphate as the phosphate donor

ABSTRACT 43K rapsyn is a peripheral protein specifically associated with the nicotinic acetylcho‐line receptor (nAChR) present in the postsynaptic membrane of the neuromuscular junction and of the electrocyte, and is essential for its clustering. Here, we demonstrate a novel specific phosphorylation of 43K rapsyn by endogenous protein kinase(s) present in Torpedo electrocyte nAChR‐rich membranes and identify thiamine triphosphate (TTP) as the phosphate donor. In the presence of Mg2+ and [γ‐32Ρ]‐TTP, 43K rapsyn is specifically phosphorylated with a 32P‐half‐maximal incorporation at ~5–25 μΜ TTP. The presence of TTP in the cytosol and of 43K rapsyn at the cytoplasmic face of the postsynaptic membrane, together with TTP‐dependent phosphor‐ylation of 43K rapsyn without added exokinases, suggests that TTP‐dependent‐43K‐rapsyn phosphor‐ylation may occur in vivo. In addition, phosphoamino acid and chemical stability analysis suggests that the residues phosphorylated are predominantly histi‐dines. Inhibition of phosphorylation by Zn2+ suggests a possible control of 43K rapsyn phosphorylation state by its zinc finger domain. Endogenous kinase(s) present in rodent brain membranes can also use [γ‐32P]‐TTP as a phosphodonor. The use of a phosphodonor (TTP) belonging to the thiamine family but not to the classical (ATP, GTP) purine triphosphate family represents a novel phosphorylation pathway possibly important for synaptic proteins.—Nghiêm, H.‐O., Bettendorff, L., Changeux, J.‐P. Specific phosphorylation of Torpedo 43K rapsyn by endogenous kinase(s) with thiamine triphosphate as the phosphate donor. FASEB J. 14, 543–554 (2000)