Worm Breeder's Gazette 15(4): 20 (October 1, 1998)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

pat-4 is an Integrin-Linked Kinase Homologue and Required for Assembly of the Myofilament Lattice in C. elegans

A. Craig Mackinnon, Jr., Benjamin D. Williams

Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, 601 South Goodwin Ave., Urbana, IL  61801

A previous genetic screen for mutants with the Pat phenotype (paralyzed, arrested elongation at two-fold) yielded a subset of mutants displaying severely disrupted sarcomere assembly in embryonic body wall muscle cells (Williams, 1994). Mutants in this subset have (1) defined the pat-2 and pat-3 genes which code for the only alpha- and beta-integrin subunits identified in C. elegans body wall muscle cells to date, (2) provided the first loss-of-function allele for the unc-52 gene which encodes perlecan, a potential ligand for the putative PAT-2/ PAT-3 integrin heterodimer, and (3) identified several new genes likely to code for proteins that work in conjunction with integrin during muscle cell differentiation. By using a candidate gene/ positional cloning approach, we have determined the molecular identity of one of these newly defined genes, pat-4.

We genetically mapped pat-4 to the left end of chromosome III which has recently been sequenced by the Genome Sequencing Consortium. This region contains two promising pat-4 candidates: the predicted genes K10F12.3 and C29F9.7 which are homologues of phospholipase C (PLC) and integrin-linked kinase (ILK), respectively. Vertebrate PLC is known to translocate to the actin cytoskeleton following integrin activation (Banno, 1996) and may be involved in the regulation of cytoskeletal architecture. We were unable to rescue pat-4 animals when we tested multiple extrachromosomal arrays (n=8) containing C17C2, a cosmid which includes the predicted gene K10F12.3, nor were we able to rescue pat-4 when we injected a genomic fragment containing only K10F12.3 plus 3.5kb of upstream sequence. Vertebrate ILK was recently shown to bind the cytoplasmic tail of the integrin beta subunit (Hannigan, 1996). In addition, ILK has been shown to play a role in ECM assembly (Wu, 1998), as well as, beta-catenin and LEF-1 signaling pathways (Novak, 1998). We rescued pat-4(st551) animals with a 7.7 kb PCR fragment containing C29F9.7 plus 2.0kb of upstream sequence and no other predicted genes. Furthermore, we prepared dsRNA from the full length cDNA clone yk199a2 (obtained from Yuji Kohara) and injected it into the gonads of N2 hermaphrodites. In the F1 brood, 98% (n=2000) of the animals arrested with the Pat phenotype, suggesting further that pat-4 corresponds to C29F9.7 and that Pat is the loss of function phenotype. Together, these results indicate that pat-4 codes for the C. elegans ILK, and ILK is therefore required for assembly of the myofilament lattice in bodywall muscle.

An amino acid alignment between the predicted PAT-4 sequence and the sequence of human ILK reveals that they are 77% similar (58% identical). Particularly striking is the presence of 4 adjacent ankyrin repeats (Lux, 1990) found in the N-terminal half of the polypeptide. These repeats comprise the motif -G-TPLH-AA-GH-V/A--LL--GA--N/D---- and are thought to mediate protein-protein interactions. The C-terminal half of human ILK and the predicted sequence of PAT-4 are homologous with protein kinase subdomains I to XI (Hanks, 1988), and the integrin binding site of human ILK maps to this region. Interestingly, kinase subdomain VII, which is a DFG triplet involved in ATP binding, is absent in both human ILK and PAT-4. Instead, both molecules contain a highly conserved 10 amino acid motif which may be functionally significant.

Currently, we are sequencing the four pat-4 mutant alleles revealed in our previous genetic screen and examining the localization of PAT-4 in developing muscle cells. The finding that pat-4 encodes an ILK homologue suggests that integrin mediated signaling events are critical for cytoskeletal assembly during muscle cell development.