title: 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.
abstract: The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14. 17.3) contains multiple signals determining its subcellular localization. Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase. In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic. P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D. Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2. Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted. P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein.
authors.affiliation: Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore Maryland 21205, USA.
authors.forename: B D
authors.initials: BD
authors.lastname: Caldwell
authors.forename: D N
authors.initials: DN
authors.lastname: Darlington
authors.forename: P
authors.initials: P
authors.lastname: Penzes
authors.forename: R C
authors.initials: RC
authors.lastname: Johnson
authors.forename: B A
authors.initials: BA
authors.lastname: Eipper
authors.forename: R E
authors.initials: RE
authors.lastname: Mains
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D047908:Intracellular Signaling Peptides and Proteins; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C102364:Uhmk1 protein, rat; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases