Cysteine-rich Protein 2, a Novel Substrate for cGMP Kinase I in Enteric Neurons and Intestinal Smooth Muscle

Winfried L. Neuhuber,Norbert Klugbauer,Hans-Dieter Allescher,Peter Ruth,Andrea Huber

doi:10.1074/jbc.275.8.5504

Abstract

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.

Highlights

Nitric oxide/cGMP/cGMP kinase I signaling causes relaxation of intestinal smooth muscle
1 The abbreviations used are: cGKI, cGMP-dependent protein kinase I; CRP2, cysteine-rich protein 2; GST, glutathione S-transferase; MES, cGKI, cGKI␣ and cGKI␤, show a tissue-specific distribution [15], with cGKI␤ being the predominant isoform in the small intestine [5]
Identification of CRP2 as Interactor of cGKI␤ in Yeast—To identify proteins of enteric neurons and intestinal smooth muscle cells interacting with cGKI␤, a two-hybrid screen was performed using cGKI␤ as a bait and a cDNA library of the longitudinal muscle with attached myenteric plexus of the rat small intestine

Summary

Introduction

Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. 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.

Results

Conclusion