TGF-b family of growth factors regulate many aspects of cellular function. The signaling is common in diverse animal species from vertebrates to C. elegans. We have been interested in the signaling pathway of the TGF-b family. We reported about cloning of a full-length cDNA (0.9 kb) of CeBRAM-2, a
daf-1 binding protein (abstract, p 250), at the international C. elegans meeting in 1997. Briefly, CeBRAM-2 has a 57% amino acid identity over the carboxy-terminal 60 amino acids of human BRAM2 (BMP receptor associated molecule) identified by yeast two-hybrid system. HA-tagged CeBRAM-2 transiently expressed in COS7 cells was found to bind typeI receptors, both ALK-3 (mammalian) and DAF-1 (C. elegans). CeBRAM-2;;GFP fusion protein was found to be expressed dominantly in amphid and pasmid neurons (e.g. ASI, ASK, ASJ, etc.). This expression pattern was similar to that of
daf-1 (C. Gunther and D. Riddle, pers. comm.). Subsequently, the null mutant was created by Tc-1 deletion method. However, phenotype of the CeBRAM2 single mutant was not obvious. We report here the genetic interaction of the CeBRAM-2 null mutant with mutants in
daf-7 TGF-b pathway by examining dauer formation of the double mutants. The result suggests that CeBRAM-2 is involved in the
daf-7 TGF-b pathway in C elegans as a negative regulator. 20C 23C 25C genotype + CeBRAM2 + CeBRAM2 + CeBRAM2
daf-1 m40 14(137) 2(373) 66(185) 10(348) 100(317) 51(495)
m402 61(144) 6(231) 94(220) 24(329) 100(481) 72(750)
m213 92(318) 27(245) 95(440) 37(308) 100(614) 80(482)
daf-7 e1372 60(210) 4(316) 98(295) 52(261) 100(493) 89(335) daf-11m47 8(154) 5(281) N.D. N.D. 56(1488) 16(2323) daf-14m77 7(247) 3(492) N.D. N.D. 47(1283) 38(1260) TABLE Percent dauer formation of CeBRAM2 and daf-c double mutants at various temperatures .The percentage of animals forming dauers is given, with the total number of animals counted given in parentheses. + indicate the daf-c single mutants, and CeBRAM indicates daf-c;CeBRAM-2 double mutants. These results show that CeBRAM-2 mutant has a week suppressor of DAF-c phenotype. CeBRAM-2 was not suppressed
daf-14 SMAD, so CeBRAM-2 is hypostatic to SMAD, and epistatic to
daf-7 TGF-b ligand and
daf-1 type I receptor. Rescue experiment was done with a CeBRAM-2 genomic fragment, to
daf-1 (
m40);CeBRAM-2 double mutant. Approximately 80 % of transgenic progeny formed dauers at 25!C, suggesting that suppression of dauer formation in the double mutant was recovered. Together, we hypothesize that CeBRAM-2 functions as a negative regulator by binding TGF-b type I receptors like as FKBP12 (Y., Chen, J., Massague, EMBO J. 16 3866-3876, 1997).