Purification, cloning, and expression of a novel, endogenous, calcium-sensitive, 28-kDa phosphoprotein.
In gastric parietal cells, cholinergically induced increases in intracellular free calcium concentrations have been well characterized, but little is known about the signaling events beyond the initial rise in intracellular calcium. In the present study, we report the isolation of a 28-kDa protein, which is rapidly phosphorylated in intact, enriched parietal cells in response to both the cholinergic agonist, carbachol, and the calcium ionophore, ionomycin. A combination of in situ 32P labeling and one- and two-dimensional gel electrophoresis was used to acquire sufficient quantities of protein to obtain partial amino acid sequence. Cloning of the pp28 cDNA revealed a novel protein which we have named CSPP28 based on its calcium-sensitive phosphorylation. There are three CSPP28 mRNA species (1.7, 2.2, and 3.3 kilobases) that are widely distributed throughout a variety of rabbit tissues. Recombinant CSPP28 was phosphorylated by both crude parietal cell homogenate and purified CaM kinase II in a calcium/calmodulin-dependent manner. We propose that CSPP28 may play an important and ubiquitous role in the calcium signaling pathway.
Parente
J A
JA
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Goldenring
J R
JR
Petropoulos
A C
AC
Hellman
U
U
Chew
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins; D010277:Parasympathomimetics / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000235:genetics*; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013270:Stomach / Q000737:chemistry
C101097:CSPP28 protein, Oryctolagus cuniculus; D017931:DNA Primers; D009363:Neoplasm Proteins; D010277:Parasympathomimetics; D010446:Peptide Fragments; D010750:Phosphoproteins; D002217:Carbachol; D002118:Calcium