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.
cki-2 is a previously uncharacterised C. elegans CIP/KIP family homologue. Ectopically expressed CKI-2 colocalizes with chromosomal DNA resulting in a penetrant embryonic lethality. Little, however, is known about the loss-of-function phenotype of
cki-2. Using a post-transcriptional silencing approach we have found that
cki-2 co-suppressed embryos arrest at an early developmental stage with a highly disorganized intestinal morphology and a substantially increased number of intestinal and pharyngeal cells, suggesting that
cki-2 may regulate embryonic cell division in these lineages. Alternatively,
cki-2 may affect patterning more indirectly by altering the replication timing. In addition to its potential mitotic role in the embryo,
cki-2 co-suppression also causes an embryonic arrest at the one cell stage. Our observations suggest that
cki-2 may have a novel meiotic role, where
cki-2 co-suppressed embryos show apparent meiotic defects including extra polar body formation and aberrant polar body extrusion in addition to defects in pronuclear fusion and subsequent cytokinesis. Radiation sensitivity was investigated in
cki-2 co-suppresssed embryos which showed an increased sensitivity to ionizing radiation. Our observation suggests that
cki-2 may therefore be involved in a DNA damage checkpoint pathway. We are presently investigating the radiation sensitivity of the
cki-2 co-suppressed animals in different genetic combinations, which may allow us to establish a connection between
cki-2 and this pathway. Through yeast two-hybrid screens we found that CKI-2 interacts with PCN-1 (PCNA orthologue), RNF-1 (RING domain protein), and SMO-1 (SUMO orthologue). Both CKI-2 and RNF-1 showed a strong and specific interaction with SMO-1. Our in vivo competition assays reveal that SMO-1 antagonizes the CKI-2/RNF-1 interaction and that the CKI-2 N-terminal domain, which contains consensus SMO-1 conjugation sites, is required for this antagonism. Genetic interactions of
cki-2 and its interacting partners further reveal that both
rnf-1 and
smo-1 act as negative regulators of
cki-2. We further showed that CKI-2 can be modified by mono-ubiquitination in vivo therefore adding another level of complexity to the regulation of CKI-2. We are presently investigating whether CKI-2 is modified by SMO-1 in vivo. We are currently characterizing how these interacting partners of CKI-2 regulate its function during C. elegans development and we believe that our work underscores a novel regulatory mechanism for CIP/KIP family proteins.