The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase

The cytosolic domain of the peptide-processing integral membrane protein pe ptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) contains mu ltiple signals determining its subcellular localization. Three PAM cytosoli c interactor proteins (P-CIPs) were identified using the yeast two hybrid s ystem (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Main s, R, E., and Eipper, B, A. (1996) J. Biol. Chem. 271, 28636-28640); the pa rtial amino acid sequence of P-CIP2 suggested that it was a protein kinase, In situ hybridization and immunocytochemistry show that P-CIP2 is expresse d widely throughout the brain; PAM and P-CIP2 are expressed in the same neu rons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is most ly cytosolic P-CIP2 is a highly selective kinase, phosphorylating the cytos olic domain of PAM, but not the corresponding region of furin or carboxypep tidase D, Although P-CIP2 interacts with stathmin, it does not phosphorylat e stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosp horylated by P-CIP2, Based on both in vitro binding experiments and co-immu noprecipitation from cell extracts, P-CIP2 Interacts with PAM proteins cont aining the wild type cytosolic domain, but not with mutant forms of PAM who se trafficking is disrupted. P-CIP2, through its highly selective phosphory lation of a key site in the cytosolic domain of PAM, appears to play a crit ical role in the trafficking of this protein.