A hyaluronate binding protein transiently codistributes with p21 in cultured cell lines

Hyaluronate (HA) affects the migratory and adhesive properties of cells. HABP, one of the sites which bind HA, localizes in the ruffling lamellae of normal migrating fibroblasts. Similarly, p21 in K-ras oncogene-transformed cells appears enhanced in membrane ruffles. To investigate the possibility that p21 and HABP are functionally linked, their subcellular distribution in two K-ras-transformed lines was examined by double label immunofluorescence and correlated with motility. In both lines, the majority of cells were p21^k-ras and HABP positive at 24 h after subculture. However, immunofluorescence for HABP both decreased and relocated, from ruffles and cell processes to cell bodies, with time whereas the intensity and distribution of staining for p21 remained constant. In doubly positive cells, HABP and p21 colocalized in the ruffles at 24 h, but not at 72 h after subculture. The times after subculture at which changes in the immunofluorescent pattern of HABP occurred differed with cell type and correlated with their migratory rate. Thus, the migratory rate of KNRK cells, which was less than in the K-C3H-10T1/2 cells, correlated with both an earlier decrease in HABP and an earlier loss of codistribution between HABP and p21 compared to K-C3H-10T1/2 cells. Further evidence of a functional link between HABP and p21^k-ras was suggested by the ability of hyaluronic acid, which induces ruffling in K-C3H-10T1/2 cells, to promote the coassociation of p21^k-ras and HABP. These results demonstrate a transient codistribution of p21 and HABP, in ruffles, that is possibly related to migratory activity and/or cell-surface changes following subculture.