We have used an affinity purification method to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown here to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage.

A proline-directed serine/threonine ceramide-activated protein (CAP) kinase mediates transmembrane signaling through the sphingomyelin pathway. CAP kinase reportedly initiates proinflammatory TNF alpha action by phosphorylating and activating Raf-1. The present studies delineate kinase suppressor of Ras (KSR), identified genetically in Caenorhabditis elegans and Drosophila, as CAP kinase. Mouse KSR, like CAP kinase, renatures and autophosphorylates as a 100-kDa membrane-bound polypeptide. KSR overexpression constitutively activates Raf-1. TNF alpha or ceramide analogs markedly enhance KSR autophosphorylation and its ability to complex with, phosphorylate, and activate Raf-1. In vitro, low nanomolar concentrations of natural ceramide stimulate KSR to autophosphorylate, and transactivate Raf-1. Other lipid second messengers were ineffective. Moreover, Thr269 the Raf-1 site phosphorylated by CAP kinase, is also recognized by KSR. Thus, by previously established criteria, KSR appears to be CAP kinase.

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

The sphingomyelin pathway, initiated by hydrolysis of sphingomyelin to ceramide and stimulation of a Ser/Thr ceramide-activated protein (CAP) kinase, mediates tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta action. CAP kinase is membrane-bound and proline-directed, recognizing the minimal substrate motif Thr-Leu-Pro. TNF may use the sphingomyelin pathway to signal Raf1 to activate the MAP kinase cascade. Evidence shows that cytoplasmic Raf1 binds to GTP-ras upon cellular stimulation, is recruited to the plasma membrane, and activated. How membrane-bound Raf1 is activated is uncertain, but regulation of its kinase activity may involve its phosphorylation. Specific Raf kinases, however, have not hitherto been identified. Here we report that CAP kinase phosphorylates Raf1 on Thr 269, increasing its activity towards MEK (MAP kinase or ERK kinase). Moreover, in intact HL-60 cells, CAP kinase complexes with Raf1 and, in response to TNF and ceramide analogues, phosphorylates and activates Raf1, implicating CAP kinase as a link between the TNF receptor and Raf1.

Prior investigations document that proliferative signaling cascades, under some circumstances, initiate apoptosis, although mechanisms that dictate the final outcome are largely unknown. In COS-7 cells, ceramide signals Raf-1 activation through Ras

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Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP's function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP's ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP's function.

We have used single-cell photometry to measure intracellular pH (pHi) for several MDR cell lines constructed by stably transfecting LR73 chinese hamster ovary fibroblasts with mutant and wild type murine MDR 1 genes. In addition, plasma membrane electrical potential (delta psi) has been measured for the same cells by the K+/valinomycin null point titration method using the ratiometric styryl probe di-4-ANEPPS. Both the untransfected, parental cell line and a cell line expressing substantial mutant MDR 1 protein (K432R/K1074R) that is unable to confer the MDR phenotype are found to have delta psi > or = -40 (+/- 5) mV and pHi < or = 7.16 (+/- 0.03) units. In contrast, MDR cell lines constructed by transfecting wild type mu MDR 1 cDNA are found to exhibit delta psi from 15 to 19 mV lower and pHi from 0.13 to 0.34 units higher. A cell line that overexpresses crippled MDR protein (S941F) that is not resistant to colchicine or doxorubicin, but which is resistant to vinblastine [Gros, P., Dhir, R., Croop, J., & Talbot, F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7289-7293], exhibits elevated pHi and slightly elevated delta psi, relative to LR73. Northern and western blot analyses confirm the substantial overexpression of the mu MDR genes and proteins in these lines, as well as the mild overexpression of endogenous hamster p-GP mRNA in some lines. In general agreement with previous studies that examined myeloma cells overexpressing hu MDR 1 protein [Roepe, P.D., Wei, L.-Y., Cruz, J., & Carlson, D. (1993) Biochemistry 32, 11042-11056] we find that overexpression of wild type mu MDR 1 protein inhibits Cl(-)- and -HCO3-dependent pHi homeostasis. Via single-cell photometry studies we now conclude that this is due to inhibition of Na(+)-independent Cl-/-HCO3 exchange (strict anion exchange or AE). As concluded previously for other MDR cells, decreased AE activity is not due to decreased expression of the exchanger; in fact, again similar to previous work [Roepe et al. (1993) Biochemistry 32, 11042-11056], we find increased levels of AE mRNA in some MDR cell lines. Models that may explain these data that are also consistent with the known physiology of cells that endogenously express MDR protein are suggested. These data are consistent with a model for MDR protein function wherein overexpression of the protein decreases delta psi and/or elevates pHi via Cl(-)- and -HCO3-dependent mechanisms.