Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis.

In many mammalian cell types, integrin-mediated cell-matrix adhesion is required for the G1-S transition of the cell cycle. As cells approach mitosis, a dramatic remodeling of their cytoskeleton accompanies dynamic changes in matrix adhesion, suggesting a mechanistic link. However, the role of integrins in cell division remains mostly unexplored. Using two cellular systems, we demonstrate that a point mutation in the beta1 cytoplasmic domain (beta1 tail) known to decrease integrin activity supports entry into mitosis but inhibits the assembly of a radial microtubule array focused at the centrosome during interphase, the formation of a bipolar spindle at mitosis and cytokinesis. These events are restored by externally activating the mutant integrin with specific antibodies. This is the first demonstration that the integrin beta1 tail can regulate centrosome function, the assembly of the mitotic spindle, and cytokinesis.

Regulation of the catalytic activity of PTP1B: roles for cell adhesion, tyrosine residue 66, and proline residues 309 and 310.

The reversible phosphorylation of proteins on tyrosine residues is fundamental to a variety of intracellular signaling pathways and is controlled by the actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). While much progress has been made in understanding the regulation of PTKs, there is still relatively little known concerning the regulation of PTPs. Using immune complex phosphatase assays, we demonstrated that the enzymatic activity of the nonreceptor type PTP, PTP1B, is regulated by cell adhesion. Placing primary human foreskin fibroblasts (HFFs) in suspension leads to a distinct increase in PTP1B activity, whereas the readhesion of suspended HFFs onto fibronectin or collagen I inhibited activity. To gain insight into the mechanisms involved, we analyzed recombinant forms of PTP1B mutated at potential regulatory sites. Our results indicated that tyrosine residue 66 is essential for maintaining activity at 37 degrees C. We also found that the C-terminal region of PTP1B and localization to the endoplasmic reticulum are not required for the inhibition of activity by cell adhesion. However, analysis of PA-PTP1B, in which alanines are substituted for prolines 309 and 310, revealed an important role for these residues as the catalytic activity of this mutant did not decrease following readhesion onto collagen I. Since the binding of p130cas and Src to PTP1B is dependent upon these proline residues, we assayed the regulation of PTP1B in mouse embryo fibroblasts deficient in these proteins. We found that neither p130cas nor Src is required for the inhibition of PTP1B activity by adhesion to extracellular matrix proteins. Additionally, pretreatment with cytochalasin D did not prevent the reduction of PTP1B activity when cells adhered to collagen I, indicating that cell spreading is not required for this regulation. The control of the catalytic activity of PTP1B by cell adhesion demonstrated in this study is likely to have important implications for growth factor and insulin signaling.

Regulation of the association of alpha 6 beta 4 with vimentin intermediate filaments in endothelial cells.

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha 6 beta 4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha 6 beta 4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin IN VITRO: the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.