In response to αβ1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require αβ1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of αβ1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL–PIX–PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.
Visiting arrangements should reflect the needs and expectations of three very diverse groups of people, namely, patients, visitors and nursing staff. In an attempt to satisfy the demands of each group, a scheme of open visiting was introduced on a Royal Victoria Hospital ward. Hilary Beggs’ study is an investigation and evaluation of how effective the change in visiting arrangements has been, and what further improvements are necessary.
AbstractTo examine a role for focal adhesion kinase (FAK) in cardiac morphogenesis, we generated a line of mice with a conditional deletion of FAK in nkx2-5-expressing cells (herein termed FAKnk mice). FAKnk mice died shortly after birth, likely resulting from a profound subaortic ventricular septal defect and associated malalignment of the outflow tract. Additional less penetrant phenotypes included persistent truncus arteriosus and thickened valve leaflets. Thus, conditional inactivation of FAK in nkx2-5-expressing cells leads to the most common congenital heart defect that is also a subset of abnormalities associated with tetralogy of Fallot and the DiGeorge syndrome. No significant differences in proliferation or apoptosis between control and FAKnk hearts were observed. However, decreased myocardialization was observed for the conal ridges of the proximal outflow tract in FAKnk hearts. Interestingly, chemotaxis was significantly attenuated in isolated FAK-null cardiomyocytes in comparison to genetic controls, and these effects were concomitant with reduced tyrosine phosphorylation of Crk-associated substrate (CAS). Thus, it is possible that ventricular septation and appropriate outflow tract alignment is dependent, at least in part, upon FAK-dependent CAS activation and subsequent induction of polarized myocyte movement into the conal ridges. Future studies will be necessary to determine the precise contributions of the additional nkx2-5-derived lineages to the phenotypes observed. SUPPLEMENTAL MATERIALWe thank Kathleen Caron, Nadia Malouf, and Kumar Pandya (UNC-CH) for helpful advice and discussions during the preparation of the manuscript. The MF20 hybridoma antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.This work was supported in part by grants from NIH-NHLBI (HL-081844 and HL-071054) and the American Heart Association (AHA) (0355776U) to J.M.T., NIH-NHLBI (HL070953) and AHA (0555476U) to C.P.M., NS19090 to L.F.R., and HL71266 and HL49277 to O.S. J.W.H. was supported by AHA Fellow to Faculty Transition Award 0275023N.
Cancer cells require sustained oncogenic signaling in order to maintain their malignant properties. It is, however, unclear whether they possess other dependencies that can be exploited therapeutically. We report here that in a large fraction of human breast cancers, the gene encoding focal adhesion kinase (FAK), a core component of integrin signaling, was amplified and FAK mRNA was overexpressed. A mammary gland-specific deletion of Fak in mice did not seem to affect normal mammary epithelial cells, and these mice were protected from tumors initiated by the polyoma middle T oncoprotein (PyMT), which activates Ras and PI3K. FAK-deficient PyMT-transformed cells displayed both growth arrest and apoptosis, as well as diminished invasive and metastatic capacity. Upon silencing of Fak, mouse mammary tumor cells transformed by activated Ras became senescent and lost their invasive ability. Further, Neu-transformed cells also underwent growth arrest and apoptosis if integrin beta4-dependent signaling was simultaneously inactivated. Human breast cancer cells carrying oncogenic mutations that activate Ras or PI3K signaling displayed similar responses upon silencing of FAK. Mechanistic studies indicated that FAK sustains tumorigenesis by promoting Src-mediated phosphorylation of p130Cas. These results suggest that FAK supports Ras- and PI3K-dependent mammary tumor initiation, maintenance, and progression to metastasis by orchestrating multiple core cellular functions, including proliferation, survival, and avoidance of senescence.
Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59fyn in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59fyn from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59fyn did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60c-src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59fyn was constitutively associated with NCAM140, the focal adhesion kinase p125fak, a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59fyn complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125fak and p59fyn. These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125fak, and p59fyn and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration. Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59fyn in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59fyn from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59fyn did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60c-src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59fyn was constitutively associated with NCAM140, the focal adhesion kinase p125fak, a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59fyn complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125fak and p59fyn. These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125fak, and p59fyn and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.
