The long term goal of this project is to characterize the structure and function of P-57, an abundant, neurospecific calmodulin (CaM) binding protein that was discovered in this laboratory. We have purified this protein to homogeneity, determined a significant percentage of its primary structure and isolated a cDNA encoding for greater than 95% of its sequence. Furthermore, the CaM binding domain of P-57 has been identified as a nine amino acid sequence near the N-terminus. In contrast to other CaM binding proteins, P-57 has higher or equivalent affinity for CaM in the absence of Ca2+ compared to the presence of Ca2+ and it is phosphorylated by protein kinase C. Phosphorylated of P-57 by protein kinase C lowers its affinity for CaM. We hypothesize that P-57 may function to bind and localize CaM at specific sites within the cell, and that phosphorylation of P-57 and increases in free Ca2 occurring with cell stimulation may release CaM locally near its target enzymes. We propose to determine the complete amino sequence of bovine brain P-57 by conventional sequencing techniques. We will isolate a full length cDNA for P-57 and systematically modify the CaM binding domain by oligonucleotide directed mutagenesis to characterize structural elements that contribute to the unusual CaM binding properties of P-57 and construct a cDNA encoding for a P- 57 in which its CaM binding domain has been replaced by the CaM binding domain of myosin light chain kinase.
Other specific aims i nclude a search for proteins in brain that may interact with P-57 and screening of various single cell culture systems for P-57. We also propose to characterize phosphorylation of P-57 and identify the single phosphorylation site in the protein.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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University of Washington
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Liu, Y; Fisher, D A; Storm, D R (1994) Intracellular sorting of neuromodulin (GAP-43) mutants modified in the membrane targeting domain. J Neurosci 14:5807-17
Liu, Y; Fisher, D A; Storm, D R (1993) Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis. Biochemistry 32:10714-9
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Liu, Y C; Storm, D R (1991) Expression of a neuromodulin-beta-galactosidase fusion protein in primary cultured neurons and its accumulation in growth cones. Mol Cell Biochem 104:29-34
Apel, E D; Byford, M F; Au, D et al. (1990) Identification of the protein kinase C phosphorylation site in neuromodulin. Biochemistry 29:2330-5

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