Drosophila neurocalcin, a fatty acylated, Ca2+-binding protein that associates with membranes and inhibits in vitro phosphorylation of bovine rhodopsin.
Neurocalcins belong to a family of neuronal specific EF hand Ca2+-binding proteins defined by recoverin. Previously, we reported the cloning and initial characterization of neurocalcin in Drosophila melanogaster (Teng, D. H.-F., Chen, C.-K., and Hurley, J. B. (1994) J. Biol. Chem. 269, 31900-31907). We showed that the Drosophila neurocalcin protein (DrosNCa) is expressed in neurons and that bacterially expressed recombinant DrosNCa (rDrosNCa) can be myristoylated. Here, we present two lines of evidence that DrosNCa is fatty acylated in vivo. First, the mobility of affinity-purified native DrosNCa on two-dimensional gel electrophoresis is identical to that of myristoylated rDrosNCa and distinct from that of nonacylated rDrosNCa. Second, the membrane binding properties of native DrosNCa are similar to those of myristoylated rDrosNCa; both of these proteins bind to membranes at 0.2 mM Ca2+, whereas nonacylated rDrosNCa always remains soluble. It has been shown that recoverin inhibits the phosphorylation of rhodopsin when Ca2+ is present (Kawamura et al., 1993) and that a dependent recoverin/rhodopsin kinase interaction underlies the inhibitory effect of recoverin (Chen et al., 1995). Given the similarities between recoverin and neurocalcin, we examined the effect of DrosNCa on rhodopsin phosphorylation. We find that rDrosNCa is capable of inhibiting bovine rhodopsin phosphorylation in vitro in a Ca2+-dependent manner. The inhibitory activity of rDrosNCa is enhanced by myristoylation, and the potency of its effect is similar to that of recoverin. Two other related EF hand proteins, guanylate cyclase-activating protein-2 and calmodulin, are only poor inhibitors in these phosphorylation assays. in vitro inhibition of rhodopsin phosphorylation therefore appears to be an assayable property of a subset of recoverin-like proteins.
Faurobert
E
E
Howard Hughes Medical Institute and the Department of Biochemistry, University of Washington, Box 357370, Seattle, Washington 98195, USA.
Chen
C K
CK
Hurley
J B
JB
Teng
D H
DH
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002451:Cell Compartmentation; D004331:Drosophila melanogaster / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D008970:Molecular Weight; D009226:Myristates; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051596:Neurocalcin; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins; D012243:Rhodopsin / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D002135:Calcium-Binding Proteins; D009226:Myristates; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins; D014364:Tryptophan; D012243:Rhodopsin; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D002118:Calcium