No abstract

The transcriptional activator nuclear factor kappa B (NF-B) is required for the upregulation of a large number of genes in response to inflammation, viral and bacterial infection, and other stress stimuli. Genes that respond to NF-B encode a variety of cytokines, cell adhesion molecules, and acute-phase response proteins as well as apoptotic suppressor and effector proteins. It is believed that this reprogramming of gene expression is essential for cell survival during situations of physiological crisis (61). The activation of NF-B in response to stimulation by the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin 1 beta (IL-1␤) has been extensively studied (17, 30); however, the mechanisms that modulate and eventually limit these responses are still poorly understood (61).We report here that the recently discovered protein kinase inhibitor protein RKIP (Raf kinase inhibitor protein) acts to inhibit NF-B activation. RKIP was first identified as an interacting partner of Raf-1 and shown to function as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by 76). The Raf-1-initiated pathway is comprised of three sequentially acting protein kinases: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAPK. This basic relationship has now been found to be conserved in several protein kinase pathways. In the Raf-1 pathway the MAPK is ERK1/2 (extracellular signal-regulated kinase 1 and 2), the MAPKK is MEK1 (MAP/ERK kinase 1), and the MAPKKK is Raf-1 itself. Functional studies using both gain-of-function and loss-of-function approaches demonstrated that RKIP disrupts the interaction between 76). Depletion of endogenous RKIP upregulated Raf-1 kinase activity and MAPK signaling, whereas ectopic expression of RKIP suppressed Raf-1 kinase activity and MAPK signaling as well as v-Raf-mediated transformation. Biochemical studies showed that RKIP efficiently dissociated preformed Raf/MEK complexes and behaved kinetically as a competitive inhibitor of MEK phosphorylation. In vivo, the association of endogenous RKIP with Raf-1 correlated inversely with Raf-1 kinase activity during serum stimulation of quiescent cells.Active NF-B is a dimer that can be assembled from several members of the Rel family of transcription factors, and some form of NF-B is expressed in most cell types (61). In unstimulated cells, NF-B is retained in the cytoplasm in an inactive form bound to a family of inhibitory proteins known as IB (inhibitors of B). Activation of NF-B requires the phosphorylation and degradation of IB, which allows the NF-B dimer to translocate into the nucleus. Virtually all of the many stimuli that can activate NF-B cause the phosphorylation of IB on

The Raf-1 kinase is regulated by phosphorylation, and Ser259 has been identi®ed as an inhibitory phosphorylation site. Here we show that the dephosphorylation of Ser259 is an essential part of the Raf-1 activation process, and further reveal the molecular role of Ser259. The fraction of Raf-1 that is phosphorylated on Ser259 is refractory to mitogenic stimulation. Mutating Ser259 elevates kinase activity because of enhanced binding to Ras and constitutive membrane recruitment. This facilitates the phosphorylation of an activating site, Ser338. The mutation of Ser259 also increases the functional coupling to MEK, augmenting the ef®ciency of MEK activation. Our results suggest that Ser259 regulates the coupling of Raf-1 to upstream activators as well as to its downstream substrate MEK, thus determining the pool of Raf-1 that is competent for signalling. They also suggest a new model for Raf-1 activation where the release of repression through Ser259 dephosphorylation is the pivotal step.

Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. (Chatterjee et al., 2004;Park et al., 2005). Consistent with its demonstrated inhibitory effect on Raf and NF-kB signaling, we and others have shown that the expression levels of RKIP are downregulated in a number of tumors, including highly metastatic prostate, breast and colon cancer, hepatocellular carcinoma, melanomas and insulinomas (Fu et al., 2003;Chatterjee et al., 2004;Schuierer et al., 2004Schuierer et al., , 2006Zhang et al., 2004;Hegan et al., 2005;Al-Mulla et al., 2006;Lee et al., 2006). The importance of RKIP in metastases was highlighted by the finding that restoration of RKIP expression inhibits prostate cancer metastasis in a murine model (Fu et al., 2003(Fu et al., , 2005. More recent studies have shown that RKIP is also a good prognostic marker of the pathogenesis of human prostate cancer (Fu et al., 2005) and a prognostic indicator for overall survival and disease-free survival in colorectal cancer (Al-Mulla et al., 2006). Collectively, these studies suggest that RKIP is a novel cancer metastasis suppressor and an effector of signal transduction pathways leading to apoptosis. In spite of the abundance of experimental evidence on the deleterious consequences of reduced RKIP expression in tumors, the mechanisms responsible for the downregulation of RKIP in cancer are not completely understood.To set up a system to study the transcription regulation of RKIP, we examined RKIP expression levels in cancer cell lines with different metastatic capacity. In accordance with clinical tumor studies, we observed that expression levels of RKIP proteins progressively decreased in breast and prostate cancer cell lines of increasing metastatic potential. The expression of RKIP is low in invasive and metastatic breast (MB231, MB435 and 578T) and prostate (DU145 and PC3) cell lines and high in noninvasive cell lines like MCF7, BT20 and LNCaP (Figure 1a). Notably, RKIP protein levels correlated well with those of the intercellular adhesion protein E-cadherin (E-cad). E-cad is a well-documented tumor metastasis suppressor protein that is regulated by the Snail and closely related Slug transcription factors (Peinado et al., 2007). Quantitation of RKIP transcript levels in the different cancer cell lines by qRT-PCR demonstrated that they correlated with the levels of the protein, (Figure 1b), suggesting that RKIP expression is downregulated at the RNA level, via changes...

The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation.The Raf-1 kinase is at the interface of a signaling pathway that connects cell surface receptors to nuclear transcription factors. Raf-1 is the entry point to the ERK/MAPK (extracellular-signal-regulated kinase/mitogen-activated protein kinase) pathway. It phosphorylates and activates MEK (MAPK/ERK kinase), which in turn phosphorylates and activates ERK. Raf-1 activation is initiated by binding of Raf-1 to GTP-loaded Ras, which results in the translocation of Raf-1 from the cytosol to the cell membrane, where activation takes place (reviewed in references 4, 18, 21, 22, and 26). Activation involves phosphorylation on serine 338 (17, 23) and tyrosine 341 (10, 23) as well as other yet-unknown modifications. It has been extremely difficult to identify the activating modifications, in part because presumably only a small fraction of Raf-1 becomes activated (14). Hence, despite receiving intensive attention during the last 10 years, several facets of Raf-1 regulation have evaded elucidation. This also pertains to the cross talk between the Raf-MEK-ERK and cyclic AMP (cAMP) signaling systems; and the mechanism of Raf-1 regulation by cAMP has not been completely clarified (15).In resting cells Raf-1 is phosphorylated on serines 43, 259, and 621 (27). Serines 43 and 621 have been previously described as target sites for the cAMP-regulated protein kinase, PKA, which inhibits Raf-1 (13, 25, 38). Phosphorylation of serine 43 was reported to diminish the affinity of Raf-1 for Ras (38) and thereby to interfere with Raf-1 activation (30, 38), although recent results dispute this (34). The role of serine 621 phosphorylation is more difficult to study, because mutation of this residue is incompatible with kinase function (25,27). This observation was taken as an indication that serine 621 phosphorylation is essential for Raf-1 function. Biochemical experiments confirmed that th...