Hypoxia/Ischemia Induces Dephosphorylation of Rat Brain Neuromodulin/GAP‐43 In Vivo

: The in vivo state of phosphorylation and the modificationof two Cys residues of neuromodulin/GAP‐43 (Nm) were analyzed by electrosprayionization‐mass spectrometry (ES‐MS). The protein was purified from rat brainwith homogenization buffer containing 1% Nonidet P‐40, protease inhibitors,protein phosphatase inhibitors, and sulfhydryl reagent, 4‐vinylpyridine. Nmwas purified by HPLC and ion‐exchange chromatography, and the variousfractions were identified by ES‐MS as unphosphorylated and mono‐, di‐, tri‐,and tetraphosphorylated species. All of these Nm species contained 2 mol ofadded 4‐vinylpyridine per mol of Nm, suggesting that that the two Cys residuesare in the reduced form in the brain. In vivo, the majority of Nm is in thephosphorylated form (~80%), of which the levels of the mono‐ and diphosphoforms are higher than those of the tri‐ and tetraphospho species. Four in vivophosphorylation sites, Ser 41 , Thr 95 , Ser 142 ,and Thr 172 , were identified by amino acid sequencing and tandemES‐MS of the peptides derived from Lys‐C endoproteinase digestion. Among thesesites, only Ser 41 is a known target of PKC, whereas the kinasesresponsible for the phosphorylation of the other three novel sites areunknown. Hypoxia/ischemia caused a preferential dephosphorylation ofSer 41 and Thr 172 , whereas Thr 95 is the least susceptible to dephosphorylation.