Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle.
Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system. The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T. (1993) FEBS Lett. 333, 51-55). Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation. Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus. The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine.
Huber
A
A
II. Medizinische Klinik und Poliklinik, Technische Universität München, D-81675 München, Germany.
Neuhuber
W L
WL
Klugbauer
N
N
Ruth
P
P
Allescher
H D
HD
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D017615:Enteric Nervous System / Q000201:enzymology*; D015723:Gene Library; D007150:Immunohistochemistry; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D009687:Nuclear Proteins; D010766:Phosphorylation; D011061:Poly A; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D009687:Nuclear Proteins; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C074370:cysteine-rich protein, mammalian; D011061:Poly A; D017346:Protein Serine-Threonine Kinases; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases