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unusual CaM binding properties of P-57 and construct a cDNA encoding for a P- 57 in which its CaM binding domain has been replaced by the CaM binding domain of myosin light chain kinase.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.
|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|
|Chapman, E R; Alexander, K; Vorherr, T et al. (1992) Fluorescence energy transfer analysis of calmodulin-peptide complexes. Biochemistry 31:12819-25|
|Chapman, E R; Estep, R P; Storm, D R (1992) Palmitylation of neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C. J Biol Chem 267:25233-8|
|Chapman, E R; Au, D; Alexander, K A et al. (1991) Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42. J Biol Chem 266:207-13|
|Apel, E D; Litchfield, D W; Clark, R H et al. (1991) Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin. J Biol Chem 266:10544-51|
|Chapman, E R; Au, D; Nicolson, T A et al. (1991) Mutagenesis of the calmodulin binding domain of neuromodulin. Prog Brain Res 89:37-44|
|Liu, Y C; Chapman, E R; Storm, D R (1991) Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons. Neuron 6:411-20|
|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|
Showing the most recent 10 out of 18 publications