The Sos1-Rac1 signaling. Possible involvement of a vacuolar H(+)-ATPase E subunit.
We have purified and identified a 32-kDa protein interacting with the Dbl oncogene homology domain of mSos1(Sos-DH) from rat brains by glutathione S-transferase-Sos-DH affinity chromatography. Peptide sequencing revealed that the protein is identical to a positive regulatory E subunit (V-ATPase E) of a vacuolar H(+)-ATPase, which is responsible for acidification of endosome and alkalinization of intracellular pH. The interaction between V-ATPase E and Sos-DH was confirmed by yeast two-hybrid assay. A coimmunoprecipitation assay demonstrated that a V-ATPase E protein physiologically bound to mSos1, and the protein was colocalized with mSos1 in the cytoplasm, as determined by immunohistochemistry. mSos1 was found in the early endosome fraction together with V-ATPase E and Rac1, suggesting the functional involvement of mSos1/V-ATPase E complexes in the Rac1 activity at endosomes. Overexpression of V-ATPase E in COS cells enhanced the ability of mSos1 to promote the guanine nucleotide exchange activity for Rac1 and stimulated the kinase activity of Jun kinase, a downstream target of Rac1. Thus, the data indicate that V-ATPase E may participate in the regulation of the mSos1-dependent Rac1 signaling pathway involved in growth factor receptor-mediated cell growth control.
Miura
K
K
Science Applications International Corporation, SAIC Frederick, Frederick, Maryland 21702, USA.
Miyazawa
S
S
Furuta
S
S
Mitsushita
J
J
Kamijo
K
K
Ishida
H
H
Miki
T
T
Suzukawa
K
K
Resau
J
J
Copeland
T D
TD
Kamata
T
T
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002846:Chromatography, Affinity; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism; D007150:Immunohistochemistry; D019476:Insect Proteins; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D020837:SOS1 Protein / Q000378:metabolism*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D019476:Insect Proteins; D020837:SOS1 Protein; C443115:V-ATPase subunit E, Manduca sexta; D025262:Vacuolar Proton-Translocating ATPases; D020830:rac1 GTP-Binding Protein