Immunoprecipitation and Western blotting revealed that actin coprecipitated with β1 integrin from quiescent HBE cells but not from MCF-7 cells. Coprecipitation of actin with β1 integrin but not with the control IgG precipitates from HBE cells indicates that association of β1 integrin with actin is not non-specific. Since actin expression in MCF-7 cells adhering to collagen IV was comparable with that in HBE cells, the result suggests that loss of linkage between β1 integrin and the actin cytoskeleton in MCF-7 cells may not be due to the absence of actin expression.
The β1 integrin subunit interacts by its cytoplasmic domain with many actin-binding proteins, such as α-actinin, talin, and vinculin, thereby forming a link between β1 integrin and the actin cytoskeleton [7
]. Immunoprecipitation followed by Western blotting revealed that only α-actinin, but not talin or vinculin, coprecipitated with β1 integrin but not with the IgG precipitates from both HBE and MCF-7 cells. This indicates the specific association between α-actinin and β1 integrin in both HBE and MCF-7 cells. Whereas comparable amounts of α-actinin and vinculin are present in HBE and MCF-7 cells and slightly reduced level of talin is seen in MCF-7 cells, the result suggests that α-actinin, but not talin or vinculin, may predominantly mediate a one-protein link between β1 integrin and actin in both HBE and MCF-7 cells and that the loss of linkage between the two proteins in MCF-7 cells may be due to the absence of a link between β1 integrin-associated α-actinin and actin.
In spite of the fact that α-actinin may directly bind with actin [15
], structural requirements for the functions of the cytoplasmic domain of β1 or β3 integrin in cell adhesion and cell spreading are strongly suggested by many investigations [19
]. In addition, there are several isoforms within the cytoplasmic domain of β1 integrin [16
]. These observations prompted us to examine which isoform of β1 integrin is expressed in MCF-7 cells. RT-PCR, using a set of primers corresponding to the sequences covering the cytoplasmic domain of β1 integrin isoform A, demonstrated that the same cDNA fragments in lengh were amplified from HBE and MCF-7 cells. Sequence analysis indicated that the cDNA fragments had the identical sequence and corresponded to sequences encoding the cytoplasmic domain of the wild-type β1A integrin. This suggests that both HBE and MCF-7 cells express the same wild-type β1 integrin and that the loss of linkage between β1 integrin and actin may not be due to the structural difference in the cytoplasmic domain of β1 integrin in MCF-7 cells from that in HBE cells.
The relevance of phosphorylation of the β subunit of integrin at threonine [19
] or tyrosine residue [23
] to the integrin functions that include cell adhesion and invasion has been documented. Therefore we next examined the phosphorylation state of β1 integrin and its relation to the linkage formation with actin. Metabolic labeling of cells with [32
P]orthophosphoric acid and Western blotting using the anti-PY antibody indicated that β1 integrin, but not α-actinin, in either HBE or MCF-7 cells was phosphorylated at least on a tyrosine residue and that there was no apparent difference in the phosphorylation state of β1 integrin between two cells. Therefore, the susceptibility of the linkage forming ability of β1 integrin with actin to PTP or PP was examined. Effects of the PTP treatment of the β1 immunoprecipitates from HBE and MCF-7 cells were contradictory; that is, coprecipitation of actin with β1 integrin from HBE cells was lost, while that of two proteins from MCF-7 cells was induced after incubation of the PTP-treated immunoprecipitates with the supernatant of the immunoprecipitates or with exogenous human platelet actin. As the reactivity of anti-PY antibody with β1 integrin from either HBE or MCF-7 cells was lost by PTP and exogenous actin coprecipitated with PTP-treated β1 integrin from MCF-7 cells, alterations in coprecipitation of actin with β1 integrin may be due to tyrosine dephosphorylation of β1 integrin and not other proteins involved in the immunoprecipitates or the supernatant of the immunoprecipitates. In addition, the result suggests that tyrosine phosphorylation is necessary for β1 integrin to link with actin in HBE cells but inhibitory for it in MCF-7 cells. Contrary to this, treatment of the β1 integrin immunoprecipitates with PP2A1
did not result in any alteration in coprecipitation of actin with β1 integrin from HBE cells but caused coprecipitation of the two proteins from MCF-7 cells. As PP2A1
did not affect tyrosine phosphorylation of β1 integrin from either HBE or MCF-7 cells and exogenous actin became coprecipitated with PP2A1
-treated β1 integrin from MCF-7 cells as endogenous actin in the supernatant, the results suggested that PP2A1
-induced coprecipitation of β1 integrin from MCF-7 cells with actin may be due to serine/threonine dephosphorylation of β1 integrin rather than other cellular proteins and that the ability of β1 integrin to link with actin in MCF-7 cells may be prevented by its serine/threonine phosphorylation. While it appears that phosphorylation at either tyrosine or serine/threonine is necessary for β1 integrin to link with actin and phosphorylation at both tyrosine and serine/threonine prevents β1 integrin from linking with actin, the linkage formation of β1 integrin with the actin cytoskeleton may be differentially regulated in MCF-7 cells as compared to HBE cells by tyrosine or serine/threonine phosphorylation of β1 integrin.
Finally we examined whether some protein kinases associate with β1 integrin in MCF-7 cells, using antibodies to several serine/threonine protein kinases. Among these, the anti-CaMKII antibody reacted with doublet of proteins with molecular mass of around 56 kDa which coprecipitated with β1 integrin from MCF-7 cells but not from HBE cells. As the antibody to CaMKII did not react with any protein in the control IgG precipitates from MCF-7 cells, association of the doublet of proteins with β1 integrin may be specific. The results demonstrating that the β1 integrin immunoprecipitates from MCF-7 cells had the kinase activity for a specific substrate peptide, Autocamtide II, under the conditions where calmodulin and inhibitor peptides of PKA and PKC were present and that this kinase activity was completely inhibited by 10 μM KN-62, a specific inhibitor of CaMKII [26
], suggest that the doublet of proteins which associate with β1 integrin in MCF-7 cells may be CaMKII isoforms. Even though the reason for an elevated level of the kinase activity of the β1 immunoprecipitates from HBE cells by KN-62 is unknown at present, it is possible to assume that some unidentified protein kinases which are responsive to KN-62 may associate with β1 integrin in HBE cells. Coprecipitation of CaMKII with β1 integrin in MCF-7 cells does not imply their direct association. As direct association between CaMKII and α-actinin has been reported by yeast two-hybrid and biochemical assays [29
], CaMKII in MCF-7 cells may bind to α-actinin, which may also bind to β1 integrin, forming a ternary complex with α-actinin and β1 integrin. Whether serine/threonine phosphorylation of β1 integrin in MCF-7 cells is regulated by the β1 integrin-associated CaMKII, or whether loss of intracellular linkage between β1 integrin and the actin cytoskeleton causes the reduced cell surface expression of β1 integrin, are areas for further investigation.