Vacuolar H+-ATPase binding to microfilaments: regulation in response to phosphatidylinositol 3-kinase activity and detailed characterization of the actin-binding site in subunit B.
Vacuolar H(+)-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49-59 of B1 and 55-65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.
Chen
Shih-Hua
SH
Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610, USA.
Bubb
Michael R
MR
Yarmola
Elena G
EG
Zuo
Jian
J
Jiang
Jin
J
Lee
Beth S
BS
Lu
Ming
M
Gluck
Stephen L
SL
Hurst
I Rita
IR
Holliday
L Shannon
LS
D008841:Actin Cytoskeleton / Q000201:enzymology*; D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001862:Bone Resorption; D002867:Chromones / Q000494:pharmacology; D003285:Contractile Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005454:Fluorescence Polarization; D006801:Humans; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010010:Osteoclasts / Q000187:drug effects / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051304:Profilins; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D025262:Vacuolar Proton-Translocating ATPases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D002867:Chromones; D003285:Contractile Proteins; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051304:Profilins; D011993:Recombinant Fusion Proteins; D025262:Vacuolar Proton-Translocating ATPases; C495738:PFN1 protein, human; C495739:Pfn1 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one