In C. elegans,
unc-89 mutants display disorganization of muscle A-bands.
unc-89 encodes 6 major polypeptides, as large as 800 kDa, composed of immunoglobulin (Ig), fibronectin type III (Fn3), SH3, DH, PH, and protein kinase domains. Several UNC-89 isoforms contain two protein kinase domains, PK1 and PK2. Molecular modeling suggests that PK1 is inactive, whereas PK2 is catalytically active. Antibodies localize the UNC-89 proteins to the M-line. To gain further insight into the function of UNC-89, we screened a yeast 2-hybrid library, using portions of UNC-89 containing PK2, as baits. The screen resulted in 42 positive clones after re-transformation. These clones identified the same gene, B0379.4. Both isoforms, B0379.4a (38 kDa) and b (55 kDa), were represented. The only recognizable domain in B0379.4 is a serine protein phosphatase. Transgenic animals were generated carrying a plasmid in which 7.5 kb of sequence upstream of the 5end of B0379.4b was fused to GFP. Expression was observed in the same set of muscles (pharyngeal, vulval, body wall) in which
unc-89 is expressed. In addition to the PK2 kinase domain, interaction with B0379.4 required both the autoinhibitory sequence lying C-terminal of the kinase domain, and the Ig and Fn3 domains lying N-terminal of the kinase domain. Moreover, when the analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1 (Ce titin), were tested, they failed to interact with B0379.4. When Fn3-Ig-PK1 was used as bait to screen a nematode M-line bookshelf that contains portions of 16 different proteins, interaction was also found with B0379.4. The requirement for interaction of PK1 and B0379.4 are the same as for interaction of PK2 and B0379.4; i.e. the whole segment, Fn3-Ig-PK1 and autoinhibitory sequence are needed. For both PK1 and PK2, only the phosphatase domain of B0379.4 is needed for interaction. When HA tagged Ig-Fn3-PK2 and myc tagged B0379.4b were expressed in the same yeast cells, co-immunoprecipitation could be demonstrated. Separate rabbit antibodies were generated that recognize either B0379.4b or B0379.4a from worm extracts of wild type, and in truncated form from two knockout mutants. By immunofluorescence microscopy, both antibodies localize to the M-line and to a portion of the I-band. The phosphatase domain of B0379.4 is most closely related to the phosphatase domains of CTD phosphatases; B0379.4 has been re-named SCPL-1. We have expressed SCPL-1 as an MBP fusion protein and demonstrated that it has phosphatase activity in vitro with properties similar to other members of the CTD phosphatase family. By RNAi, SCPL-1 knockdown results in a weak Egl phenotype; certain alleles of
unc-89 show this phenotype, as well.