Worm Breeder's Gazette 15(3): 38 (June 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.

CeBRAM-2 that binds to DAF-1 is a negative regulator of daf-7 TGF-b pathway.

Kiyokazu Morita1, Miho Shimizu2, Hiroshi Shibuya1, Naoto Ueno1

1 Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, JAPAN.E-mail: nueno@nibb.ac.jp
2 Department of Neurology #NB411 Baylor College of Medicine 

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).