SummaryMicrotubules nucleated from -tubulin ring complexes located at the centrosome regulate the localization of organelles, promote vesicular transport and direct cell migration. Although several signaling mechanisms have been identified that regulate microtubule dynamics during interphase, signaling pathways that promote microtubule nucleation remain elusive. We assayed microtubule regrowth following nocodazole washout in human fibroblasts and CHO-K1 cells adhered to fibronectin in either normal serum-free medium or the serum-free, growth-promoting medium, CCM1, which contains IGF1 and androgen, as well as other nutrients. The results indicate that integrin-mediated adhesion is not sufficient to promote rapid microtubule regrowth in either cell type. The addition of androgen, but not IGF1, for 5 minutes was sufficient to promote rapid regrowth and this occurred by a mechanism requiring the androgen receptor. Since Src is a component of the cytoplasmic androgen-receptor-signaling complex, we examined its role using Src siRNA, the Src kinase inhibitor SU6656, and the expression of a constitutively active Src mutant. The data show that Src signaling is both required and sufficient to promote rapid microtubule regrowth in cells adhered to fibronectin. Measurement of the density of microtubules close to the centrosome and the rates of GFP-EB1-labeled microtubules emanating from the centrosome indicated that Src signaling promotes microtubule nucleation. Furthermore, recovery of GFP--tubulin at the centrosome following photobleaching and measurements of endogenous -tubulin levels at the centrosome showed that androgen and Src signaling regulate the levels of centrosomal -tubulin. Thus, we propose that androgen and Src signaling regulate microtubule nucleation during interphase by promoting the centrosomal localization of -tubulin.

SummaryProtein interactions with the integrin b-subunit cytoplasmic domain (b-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of b-tails (NPIY in b1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and b-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant b1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the b1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, FilGAP DGAP , which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the b1A tail, but not by integrins containing the b1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the b1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the b1 tail downstream of integrin activation in promoting integrin function.

Background: Centrosomal microtubule nucleation is important in the formation of the microtubule cytoskeleton. Results: An integrin mutant that suppresses MEK/ERK signaling substantially reduces microtubule nucleation. Expression of activated RAF-1 restores nucleation inhibited by the mutant integrin. Conclusion: Integrins promote microtubule nucleation by regulating MEK/ERK signaling. Significance: Environmental cues provided by cell-matrix adhesion contribute to the regulation of the microtubule nucleating activity of the centrosome.Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and ␥-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin ␤1 tail and show that this mutant substantially decreases microtubule nucleation and ␥-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of ␥-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.During interphase, the microtubule cytoskeleton determines the subcellular localization of organelles, promotes vesicular transport, and directs cell migration. The centrosome is a major site for the nucleation and organization of microtubules (1-3). It includes two centrioles embedded in pericentriolar material. The assembly of the microtubule cytoskeleton is a highly regulated process initiated by nucleation. Newly nucleated microtubules become anchored at the centrosome and exhibit growth, shrinkage, and stabilization in response to extracellular signals.␥-Tubulin is essential for microtubule nucleation. It associates with other ␥ complex proteins in the cytoplasm to form ␥-tubulin ring complexes (␥-TuRCs), 2 which then accumulate at the centrosome to serve as templates for microtubule nucleation (3). NEDD1, also known as GCP-WD, is the last protein to associate with the complex and is required for its centrosomal localization (4, 5). A similar role has recently been proposed for GCP8...

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