A novel rap1/B-Raf/14-3-3 theta protein complex is formed in vivo during the morphogenetic differentiation of postmeiotic male germ cells.
The 14-3-3 family of proteins is expressed in a broad range of organisms and tissues. Based on data essentially obtained with tissue culture cells and yeast, 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways, in particular those involving the activity of the Raf family protein kinases. The 14-3-3 theta mouse isoform is expressed almost exclusively in testis and brain. In an effort to understand the function of 14-3-3 theta in testis, we sought to identify endogenous proteins that interact with 14-3-3 theta in spermatogenic cells. A recombinant 14-3-3 theta fusion protein was used in Far Western and GST pulldown assays. Here we report that 14-3-3 theta interacts in vivo and in vitro with 93- to 95-kDa B-Raf, originally described as specific of neural tissues and never reported in male germ cells. Moreover, in mouse spermatids, i.e., the haploid cytodifferentiating cells, a so far unidentified protein complex formed by endogenous Rap1/B-Raf/14-3-3 theta can be coimmunoprecipitated. The intracellular localization of endogenous 14-3-3 theta, B-Raf, and Rap1 was analyzed in distinct spermatogenic cell types and a peculiar codistribution of the three proteins was immunorevealed in differentiating spermatids. Together, these data demonstrate that a protein complex formed by endogenous Rap1, 93- to 95-kDa B-Raf, and 14-3-3 theta exists in vivo and the finding that this has been detected in cytodifferentiating, not dividing cells, strengthens the hypothesis for a role of Rap1/B-Raf-mediated signaling in cell morphogenesis and differentiation.
Berruti
G
G
Department of Biology, Milan University, Italy. berruti@mailserver.unimi.it
D048948:14-3-3 Proteins; D000818:Animals; D002454:Cell Differentiation; D008297:Male; D008540:Meiosis; D051379:Mice; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D048948:14-3-3 Proteins; D020033:Protein Isoforms; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D020827:rap1 GTP-Binding Proteins