Vinculin controls talin engagement with the actomyosin machinery

The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs

The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A.

The HSP12 gene encodes one of the two major small heat shock proteins of Saccharomyces cerevisiae. Hsp12 accumulates massively in yeast cells exposed to heat shock, osmostress, oxidative stress, and high concentrations of alcohol as well as in early-stationary-phase cells. We have cloned an extended 5'-flanking region of the HSP12 gene in order to identify cis-acting elements involved in regulation of this highly expressed stress gene. A detailed analysis of the HSP12 promoter region revealed that five repeats of the stress-responsive CCCCT motif (stress-responsive element [STRE]) are essential to confer wild-type induced levels on a reporter gene upon osmostress, heat shock, and entry into stationary phase. Disruption of the HOG1 and PBS2 genes leads to a dramatic decrease of the HSP12 inducibility in osmostressed cells, whereas overproduction of Hog1 produces a fivefold increase in wild-type induced levels upon a shift to a high salt concentration. On the other hand, mutations resulting in high protein kinase A (PKA) activity reduce or abolish the accumulation of the HSP12 mRNA in stressed cells. Conversely, mutants containing defective PKA catalytic subunits exhibit high basal levels of HSP12 mRNA. Taken together, these results suggest that HSP12 is a target of the high-osmolarity glycerol (HOG) response pathway under negative control of the Ras-PKA pathway. Furthermore, they confirm earlier observations that STRE-like sequences are responsive to a broad range of stresses and that the HOG and Ras-PKA pathways have antagonistic effects upon CCCCT-driven transcription