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Ras mutations are present in only a subset of sporadic human cutaneous squamous cell carcinomas (cSCC) even though Ras is activated in most. This suggests that other mechanisms of Ras activation play a role in the disease. The aberrant expression of RasGRP1, a guanyl nucleotide exchange factor for Ras, is critical for mouse cSCC development through its ability to increase Ras activity. However, the role of RasGRP1 in human keratinocyte carcinogenesis remains unknown. Here we report that RasGRP1 is significantly elevated in human cSCC and that high RasGRP1 expression in human primary keratinocytes triggered activation of endogenous Ras and significant morphological changes including cytoplasmic vacuole formation and growth arrest. Moreover, RasGRP1-expressing cells were autophagic as indicated by LC3-II increase and the formation of LC3 punctae. In an in vitro organotypic skin model, wild type keratinocytes generated a well-stratified epithelium, while RasGRP1-expressing cells failed to do so. Finally, RasGRP1 induced transformation-like changes in skin cells from Li-Fraumeni patients with inactivating p53 mutations, demonstrating the oncogenic potential of this protein. These results support a role for RasGRP1 in human epidermal keratinocyte carcinogenesis and might serve as an important new therapeutic target.

Synthesis of analogues of englerin A with a reduced propensity for hydrolysis of the glycolate moiety led to a compound which possessed the renal cancer cell selectivity of the parent and was orally bioavailable in mice.

Background: RSK2 alters cell migration and metastasis, but the mechanism is incompletely understood. Results: RSK2 inactivates integrins, increases filamin binding to integrin tails, alters actin distribution, increases migration, and decreases fibronectin matrix assembly. Conclusion: RSK2 mediates inactivation of integrins and thus modulates integrin functions. Significance: These findings provide a novel mechanism by which RSK2 affects migration and may lead to more selective ways to inhibit RSK2-dependent metastasis., Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.

Loss of properly regulated cell death and cell survival pathways can contribute to the development of cancer and cancer metastasis. Cell survival signals are modulated by many different receptors, including integrins. Bit-1 is an effector of anoikis (cell death due to loss of attachment) in suspended cells. The anoikis function of Bit-1 can be counteracted by integrin-mediated cell attachment. Here, we explored integrin regulation of Bit-1 in adherent cells. We show that knockdown of endogenous Bit-1 in adherent cells decreased cell survival and re-expression of Bit-1 abrogated this effect. Furthermore, reduction of Bit-1 promoted both staurosporine and serum-deprivation induced apoptosis. Indeed knockdown of Bit-1 in these cells led to increased apoptosis as determined by caspase-3 activation and positive TUNEL staining. Bit-1 expression protected cells from apoptosis by increasing phospho-IκB levels and subsequently bcl-2 gene transcription. Protection from apoptosis under serum-free conditions correlated with bcl-2 transcription and Bcl-2 protein expression. Finally, Bit-1-mediated regulation of bcl-2 was dependent on focal adhesion kinase, PI3K, and AKT. Thus, we have elucidated an integrin-controlled pathway in which Bit-1 is, in part, responsible for the survival effects of cell-ECM interactions.

The ERK MAP (mitogen-activated protein) kinase cascade modulates many cellular processes including transcription, adhesion, growth, survival, and proliferation. One target substrate of ERK involved in regulating transcription is the p90 ribosomal S6 kinase (RSK) isozyme, RSK2. Here we demonstrate that a small death effector domain-containing protein called PEA-15 binds RSK2. RSK2 and PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) co-precipitated from cells and were colocalized in the cytoplasm. Furthermore, purified PEA-15 bound in vitro translated RSK2, suggesting that these proteins interact directly. PEA-15 does not bind to RSK1 and therefore exhibits some binding specificity. RSK2 binds the COOH terminus of PEA-15 and does not interact with its NH2-terminal death effector domain. We show that this interaction has functional consequences including the inhibition of RSK2-dependent CREB transcription. PEA-15 expression also blocks histone H3 phosphorylation, an RSK2-dependent event that may contribute to effects on gene expression. These results can be attributed to two effects of PEA-15 on RSK2. First, PEA-15 blocks nuclear accumulation of RSK2 after epidermal growth factor stimulation. Second, PEA-15 inhibits RSK2 kinase activity by 50%. A mutant of PEA-15 that binds RSK2 but is localized to the nucleus had no effect on RSK2-dependent transcription. Interestingly, this mutant also did not affect RSK2 kinase activity. This may indicate that cytoplasmic retention of RSK2 is also required for PEA-15 to impair kinase activity. PEA-15 does not alter ERK phosphorylation of RSK2 and is not itself a substrate of RSK2. Hence the effects of PEA-15 on RSK2 represent a novel mechanism for the regulation of RSK2-mediated signaling.

Phospholipase D (PLD), a signal-transducing membrane-associated enzyme, is implicated in diverse processes including apoptosis, ERK activation, and glucose transport. Prior studies have identified specific PLD activators and repressors that directly regulate its enzymatic activity. Using two-hybrid screens, we have identified PEA-15 as a PLD interactor that unexpectedly functions to alter its level of expression. PEA-15 is a widely expressed death effector domain-containing phosphoprotein involved in signal transduction, apoptosis, ERK activation, and glucose transport. The PLD1-interacting site on PEA-15 consists of part of the death effector domain domain plus additional C-terminal flanking sequences, whereas the PEA-15-interacting site on PLD1 overlaps the previously identified RhoA-interacting site. PEA-15 did not affect basal or stimulated in vitro PLD1 enzymatic activation. However, co-expression of PEA-15 increased levels of PLD1 activity. This increased activation correlated with higher PLD1 protein expression levels, as marked by faster accumulation and longer persistence of PLD1 when PEA-15 was present. PEA-15 similarly increased protein expressions level of PLD2 and co-immunoprecipitated with it. These results suggest that PEA-15 may stabilize PLD or act as a PLD chaperone. The common involvement of PEA-15 and PLD in apoptosis, ERK activation, and glucose transport additionally suggests functional significance.

In early amphibian development, interactions between fibronectin and both ectoderm and mesoderm cells are critical in the progression of gastrulation movements. In the Pleurodeles waltl embryo, it has been established that ectoderm cells of the animal hemisphere organize a fibrillar-extracellular matrix containing fibronectin. Mesoderm cells migrate along the blastocoel roof using these fibronectin fibrils as substratum. Fibronectin is an adhesive glycoprotein which possesses multiple cell-binding domains. From previous studies, it is clear that amphibian ectoderm and mesoderm cells interact with fibronectin in an RGD-dependent manner, whereas the contributions of RGD-independent domains in the adhesive behaviors of gastrula cells has not been defined. To study this question, we have used bacterially expressed Pleurodeles waltl fibronectin-fusion proteins. The approach consisted of in vitro adhesion assays with either isolated cells or tissue fragments of embryos dissected at the onset of gastrulation. Tissues were obtained from regions of the embryo which represent presumptive ectoderm cells or from the dorsal-marginal zone which contains cells of the presumptive cephalic, chordal and somitic mesoderm. The results show that both the RGD-dependent and the Hep II domains of fibronectin mediate attachment and spreading of isolated cells. Both regions cooperate to control the proper expansion of a sheet of dorsal mesoderm cells. The Hep II domain promotes the migration of cells ahead of the mesoderm-cell sheet.

The V-region represents one of three alternatively spliced segments in Xenopus fibronectin. Here, we identify this V-region as binding epitope of the monoclonal antibody (MAb 6D9) that we generated against Xenopus plasma fibronectin. By the use of this antibody we obtained new results that change the present view of the fibronectin expression pattern before gastrulation: (1) The V + -fibronectin is the major isoform expressed during early development since only a single fibronectib band is found in Western blots up to tadpole stages. (2) In contrast to previously published data we demonstrate that fibronectin expression is induced by progesterone during oocyte maturation. (3) During cleavage stages the protein is stored in the cytoplasm where it is predominantly associated with plasma membranes. Immunoelectronmicroscopy reveals that V + -fibronectin is present at the surface of animal pole blastomeres and secreted into intercellular spaces. This extracellular localization of fibronectin is predominantly observed in the marginal zone, surrounding single cells of the outer cell layer baso-laterally. In the vegetal hemisphere V + -fibronectin is restricted to the cytoplasm and accumulated at plasma membranes. With the onset of gastrulation the intracellular and membrane associated fibronectin disappears and fibronectin becomes detectable at the blastocoel roof. Since reaggregation of dissociated blastula cells ws not blocked by addition of GRGDS peptide or antibodies against fibronectin, we assume that the early expression and secretion of fibronectin serves as store to allow a rapid matrix assembly with onset of mesodermal cell migration.