Y. Abuamer et al., SUBSTRATE RECOGNITION BY OSTEOCLAST PRECURSORS INDUCES C-SRC MICROTUBULE ASSOCIATION/, The Journal of cell biology, 137(1), 1997, pp. 247-258
The osteoclast is distinguished from other macrophage polykaryons by i
ts polarization, a feature induced by substrate recognition. The most
striking component of the polarized osteoclast is its ruffled membrane
, probably reflecting insertion of intracellular vesicles into the bon
e apposed plasmalemma, The failure of osteoclasts in c-src-/- osteopet
rotic mice to form ruffled membranes indicates pp60(c-src) (c-src) is
essential to osteoclast polarization. Interestingly, c-src itself is a
vesicular protein that targets the ruffled membrane, This being the c
ase, we hypothesized that matrix recognition by osteoclasts, and their
precursors, induces c-src to associate with microtubules that traffic
proteins to the cell surface, We find abundant c-src associates with
tubulin immunoprecipitated from avian marrow macrophages (osteoclast p
recursors) maintained in the adherent, but not nonadherent, state. Sin
ce the two proteins colocalize only within adherent avian osteoclast-l
ike cells examined by double antibody immunoconfocal microscopy, c-src
/tubulin association reflects an authentic intracellular event. C-src/
tubulin association is evident within 90 min of cell-substrate recogni
tion, and the event does not reflect increased expression of either pr
otein. In vitro kinase assay demonstrates tubulin-associated c-src is
enzymatically active, phosphorylating itself as well as exogenous subs
trate. The increase in microtubule-associated kinase activity attendin
g adhesion mirrors tubulin-bound c-src and does not reflect enhanced s
pecific activity. The fact that microtubule-dissociating drugs, as wel
l as cold, prevent adherence-induced c-src/tubulin association indicat
es the protooncogene complexes primarily, if not exclusively, with pol
ymerized tubulin. Association of the two proteins does not depend upon
protein tyrosine phosphorylation and is substrate specific, as it is
induced by vitronectin and fibronectin but not type 1 collagen. Finall
y, consistent with cotransport of c-src and the osteoclast vacuolar pr
oton pump to the polarized plasmalemma, the H+-ATPase decorates microt
ubules in a manner similar to the protooncogene, specifically coimmuno
precipitates with c-src from the osteoclast light Golgi membrane fract
ion, and is present, with c-src, in preparations enriched with acidify
ing vesicles reconstituted from the osteoclast ruffled membrane.