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 intes tinal smooth muscle, In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cG KI beta has been isolated from a rat intestinal cDNA library using the yeas t 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). Rec ombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKI beta into COS cells confirmed t he phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo p hosphorylation, Immunohistochemical analysis using confocal laser scan micr oscopy showed a co-localization of CRP2 and cGKI in the inner part of the c ircular 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 tha t CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the sma ll intestine.