Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK modul

Mechanism of regulation of Raf-1 by Ca2+/Calmodulin-dependent kinase II

The calcium-calmodulin dependent kinase II (CaMKII) is an ubiquitous serine/threonine protein kinase involved in multiple signalings and biological functions. It has been demonstrated that in epithelial and mesenchimal cells CaMKII participates with Ras to Raf-1 activation and that it is necessary for ERK activation by diverse factors. Raf-1 activation is complex. Maximal Raf-1 activation is reached by phosphorylation at Y341 by Src and at S338. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 is not definitively identified. Aim of my thesis is to go more insight into the molecular mechanisms of CaMKII/Raf-1 interaction and to verify the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To this purpose, I investigated the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors, in COS-7 and NIH3T3 cells. Serum, SrcY527 and RasV12 activated CaMKII. CaMKII was necessary for Raf-1 and ERK activation by all these factors. CaMKII was necessary to the phosphorylation of S338 Raf-1 by serum, fibronectin or oncogenic Ras. Conversely, the inhibition of phosphatidylinositol 3-kinase, which in turn activates Pak3, was ineffective. The direct kinase activity of CaMKII on the serine 338 residue, was demonstrated in vitro by interaction of purified kinases. These results demonstrate that CaMKII phosphorylates Raf-1 at S338 and partecipates to ERK activation upon different physiologic and pathologic stimuli in the MAPK cascade. This kinase, might have a role in cancers harbouring oncogenic Ras and could represent a new therapeutic target for pharmacological intervention in these tumors

PDE8 controls CD4(+) T cell motility through the PDE8A-Raf-1 kinase signaling complex

The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4(+) T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells, we used a cell permeable peptide that selectively disrupts the PDE8A-Raf-1 interaction. The disruptor peptide inhibits the Teff-endothelial cell interaction more potently than the enzymatic inhibitor. Furthermore, the LFA-1/ICAM-1 interaction was identified as a target of disruptor peptide mediated reduction of adhesion, spreading and locomotion of Teff cells under flow. Mechanistically, we observed that disruption of the PDE8A-Raf-1 complex profoundly alters Raf-1 signaling in Teff cells. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase complex disruptors decreases Teff cell adhesion and migration under flow, and represents a novel approach to target T cells in inflammation

The many-faced KSR1: a tumor suppressor in breast cancer

Emerging evidence supports the dual function of kinase suppressor of Ras 1 (KSR1) as an active kinase and a scaffold, although it has been extensively referred as a pseudokinase, due to the absence of key residues in its catalytic domain [1, 2]. As a scaffolding protein, KSR1 orchestrates the assembly of the protein kinases RAF, mitogen activated protein kinase (MAPK) kinase (MEK), and extracellular signal-regulated kinase (ERK) in the canonical Ras-RAF-MAPKs pathway, in a Ras-dependent manner or upon growth factor treatment [1, 3]. Conversely, structural and biochemical studies reveal that KSR1 is capable of phosphorylating MEK and more importantly, the catalytic activity of KSR is markedly increased when BRAF or inhibitor-bound CRAF is introduced in the complexes [1, 4, 5]. Such findings add complexity to th