In C. elegans, two CIP/KIP family CDK inhibitors (CKIs) are encoded by the
cki-1 and
cki-2 genes found in tandem on chromosome II. Inactivation of
cki-2 by RNAi (RNA mediated interference) or cosuppression produces an impenetrant embryonic arrest phenotype. It is unclear whether
cki-2 functions as a negative cell cycle regulator, nor is it clear whether the embryonic lethality seen in
cki-2 (RNAi) or cosuppressed worms is due to cell cycle defects. To gain further insight into
cki-2 function, we performed a yeast two-hybrid screen in order to identify interacting partners of CKI-2. We have identified three CKI-2 interactors: W03D2.4 (CePCNA), C06A5.9 (CeRING), and K12C11.2 (CeSUMO-1). To identify how these proteins interact, we performed directional yeast two-hybrid assays that showed CeSUMO-1 also interacted with CeRING. However, CeSUMO-1 did not interact with CKI-1, which implies that CeSUMO-1 regulate CKI-2 specifically. Because CeSUMO-1 interacted with both CeRING and CKI-2, and that the CePCNA binding domain is located near CeRING binding domain on CKI-2, we postulate that CeRING might antagonize the CKI-2/CePCNA interaction through some dynamic regulation potentially mediated by CeSUMO-1. This possibility was supported by competitive inhibition analysis using inducible expression of CeSUMO-1 in yeast. We showed that CeSUMO-1 antagonized the CKI-2/CeRING interaction, but not the CKI-2/CePCNA interaction. Since other known SUMO-1 binding proteins obtained from yeast two-hybrid screens have been reported to be bona fide substrates of SUMO-1 conjugation in vivo, we are investigating whether CKI-2 is also modified by CeSUMO-1 in vivo. To better understand the role of these regulatory interactions on CKI-2 function in early embryonic development, we are using a
pie-1 promoter-based germline expression system to visualize maternal CKI-2::GFP and how this may be affected in CeSUMO-1 (RNAi) worms. The nature of the interacting partners of CKI-2 suggests that these proteins regulate CKI-2 during C. elegans development through SUMOylation, which highlights a novel regulatory mechanism for CIP/KIP family proteins.