ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix.

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.

The ect2 rho Guanine nucleotide exchange factor is essential for early mouse development and normal cell cytokinesis and migration.

Ect2 is a member of the human Dbl family of guanine nucleotide exchange factors (RhoGEFs) that serve as activators of Rho family small GTPases. Although Ect2 is one of at least 25 RhoGEFs that can activate the RhoA small GTPase, cell culture studies using established cell lines determined that Ect2 is essential for mammalian cell cytokinesis and proliferation. To address the function of Ect2 in normal mammalian development, we performed gene targeting to generate Ect2 knockout mice. The heterozygous Ect2(+/-) mice showed normal development and life span, indicating that Ect2 haplodeficiency was not deleterious for development or growth. In contrast, Ect2(-/-) embryos were not found at birth or postimplantation stages. Ect2(-/-) blastocysts were recovered at embryonic day 3.5 but did not give rise to viable outgrowths in culture, indicating that Ect2 is required for peri-implantation development. To further assess the importance of Ect2 in normal cell physiology, we isolated primary fibroblasts from Ect2(fl/fl) embryos (MEFs) and ablated Ect2 using adenoviral delivery of Cre recombinase. We observed a significant increase in multinucleated cells and accumulation of cells in G2/M phase, consistent with a role for Ect2 in cytokinesis. Ect2 deficiency also caused enlargement of the cytoplasm and impaired cell migration. Finally, although Ect2-dependent activation of RhoA has been implicated in cytokinesis, Ect2 can also activate Rac1 and Cdc42 to cause growth transformation. Surprisingly, ectopic expression of constitutively activated RhoA, Rac1, or Cdc42, known substrates of Ect2, failed to phenocopy Ect2 and did not rescue the defect in cytokinesis caused by loss of Ect2. In summary, our results establish the unique role of Ect2 in development and normal cell proliferation.

Citron kinase, a RhoA effector, enhances HIV-1 virion production by modulating exocytosis.

RhoGTPases play important roles in the regulation of protein transport and membrane recycling. Little is known, however, about how RhoGTPases affect HIV-1 virion production, which is dependent on the endosomal sorting pathway. We report that ectopic expression of citron kinase (citron-K), a RhoA effector, preferentially enhances HIV-1 virion production. Depletion of endogenous citron-K inhibits HIV-1 virion production. Citron-N, which lacks the kinase domain, also enhances HIV-1 virion production. The leucine zipper, Rho-binding and zinc finger domains of citron-N are necessary for the enhancement activity. Citron-K also enhances murine leukemia virion production and the HIV-1 late domain is not required for the citron-K-mediated enhancement. Ectopic expression of citron-K leads to the formation of cytoplasmic structures containing citron-K and HIV-1 Gag proteins. HIV-1 and citron-K cooperatively enhance acidic endosome and lysosome compartments. Finally, citron-K promotes exocytosis of microvesicles or exosomes that co-purify with HIV-1 virions. We conclude that citron-K enhances HIV-1 virion production by stimulating the endosomal compartments and exocytosis.

RhoA biological activity is dependent on prenylation but independent of specific isoprenoid modification.

Recent studies showed that specific isoprenoid modification may be critical for RhoB subcellular location and function. Therefore, we determined whether the function of the highly related RhoA protein is also critically dependent on specific isoprenoid modification: (a) in contrast to observations with RhoB or Ras proteins, where farnesylated and geranylgeranylated versions showed differences in subcellular location, both prenylated versions of RhoA showed the same plasma membrane and cytosolic location; (b) a farnesylated version of activated RhoA(63L) retained the same diverse functions as the normally geranylgeranylated RhoA(63L) protein, and both proteins show indistinguishable abilities to stimulate gene expression, cause growth transformation of NIH 3T3 mouse fibroblasts, to stimulate the motility of T47D human breast epithelial cells, and to block HIV-1 viral replication and gene expression; and (c) cells expressing farnesylated RhoA retained sensitivity to the growth inhibition caused by inhibition of geranylgeranyltransferase I, indicating that other proteins are critical targets for inhibitors of geranylgeranylation.

Requirement for C-terminal sequences in regulation of Ect2 guanine nucleotide exchange specificity and transformation.

Ect2 was identified originally as a transforming protein and a member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs). Like all Dbl family proteins, Ect2 contains a tandem Dbl homology (DH) and pleckstrin homology (PH) domain structure. Previous studies demonstrated that N-terminal deletion of sequences upstream of the DH domain created a constitutively activated, transforming variant of Ect2 (designated DeltaN-Ect2 DH/PH/C), indicating that the N terminus served as a negative regulator of DH domain function in vivo. The role of sequences C-terminal to the DH domain has not been established. Therefore, we assessed the consequences of mutation of C-terminal sequences on Ect2-transforming activity. Surprisingly, in contrast to observations with other Dbl family proteins, we found that mutation of the invariant tryptophan residue in the PH domain did not impair DeltaN-Ect2 DH/PH/C transforming activity. Furthermore, although the sequences C-terminal to the PH domain lack any known functional domains or motifs, deletion of these sequences (DeltaN-Ect2 DH/PH) resulted in a dramatic reduction in transforming activity. Whereas DeltaN-Ect2 caused formation of lamellipodia, DeltaN-Ect2 DH/PH enhanced actin stress fiber formation, suggesting that C-terminal sequences influenced Ect2 Rho GTPase specificity. Consistent with this possibility, we determined that DeltaN-Ect2 DH/PH activated RhoA, but not Rac1 or Cdc42, whereas DeltaN-Ect2 DH/PH/C activated all three Rho GTPases in vivo. Taken together, these observations suggest that regions of Ect2 C-terminal to the DH domain alter the profile of Rho GTPases activated in vivo and consequently may contribute to the enhanced transforming activity of DeltaN-Ect2 DH/PH/C.