Mechanisms that regulate signal propagation through the ERK/MAPK pathway are still poorly understood. Several proteins are suspected to play critical roles in this process. One of these is Kinase Suppressor of Ras (KSR), a component previously identified in RAS-dependent genetic screens in Drosophila and Caenorhabditis elegans. Here, we show that KSR functions upstream of MEK within the ERK/MAPK module. In agreement with this, we found that KSR facilitates the phosphorylation of MEK by RAF. We further show that KSR associates independently with RAF and MEK, and that these interactions lead to the formation of a RAF/MEK complex, thereby positioning RAF in close proximity to its substrate MEK. These findings suggest that KSR functions as a scaffold that assembles the RAF/MEK functional pair.

Connector enhancer of KSR (CNK) is a multidomain-containing protein previously identified as a positive regulator of the RAS/MAPK pathway in Drosophila. Using transfection experiments and an RNAi-based rescue assay in Drosophila S2 cells, we demonstrate that CNK has antagonistic properties with respect to RAF activity. We show that CNK's N-terminal region contains two domains (SAM and CRIC) that are essential for RAF function. Unexpectedly, we also report that the C-terminal region of CNK contains a short bipartite element that strongly inhibits RAF catalytic function. Interestingly, CNK's opposite properties appear to prevent signaling leakage from RAF to MEK in the absence of upstream signals, but then transforms into a potent RAF activator upon signal activation. Together, these findings suggest that CNK not only participates in the elusive RAF activation process, but might also contribute to the switch-like behavior of the MAPK module.

B-cell chronic lymphocytic leukemia (B-CLL) is a malignant disease characterized by an accumulation of monoclonal CD5 þ mature B cells, with a high percentage of cells arrested in the G0/G1 phase of the cell cycle, and a particular resistance toward apoptosis-inducing agents. Dok1 (downstream of tyrosine kinases) is an abundant Ras-GTPase-activating protein (Ras-GAP)-associated tyrosine kinase substrate, which negatively regulates cell proliferation, downregulates MAP kinase activation and promotes cell migration. The gene encoding Dok1 maps to human chromosome 2p13, a region previously found to be rearranged in B-CLL. We have screened the Dok1 gene for mutations from 46 individuals with B-CLL using heteroduplex analysis. A four-nucleotide GGCC deletion in the coding region was found in the leukemia cells from one patient. This mutation causes a frameshift leading to protein truncation at the carboxyl-terminus, with the acquisition of a novel amino-acid sequence. In contrast to the wild-type Dok1 protein, which has cytoplasmic/ membrane localization, the mutant Dok1 is a nuclear protein containing a functional bipartite nuclear localization signal. Whereas overexpression of wild-type Dok1 inhibited PDGF-induced MAP kinase activation, this inhibition was not observed with the mutant Dok1. Furthermore the mutant Dok1 forms heterodimers with Dok1 wild type and the association can be enhanced by Lck-mediated tyrosine-phosphorylation. This is the first example of a Dok1 mutation in B-CLL and the data suggest that Dok1 might play a role in leukemogenesis.

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES ( 348 LLKAKLTDPKED 359 ) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKK␤. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions. Dok1 belongs to a family of adaptor proteins that are heavily tyrosine phosphorylated after stimulation with epidermal growth factor receptor, insulin receptor, and antigen receptors. Tyrosine phosphorylation of Dok1 also occurs in several cell lines transformed by viral oncogenes including v-Src and v-Abl, and in chronic myelogenous leukemia cells, where it is a target of p210Bcr-Abl (2, 3, 29, 52). DOK family members (Dok1 to Dok6) and insulin receptor substrates are characterized by a pleckstrin homology domain (PH) that allows anchorage to the membrane, a phosphotyrosine binding domain that is involved in protein-protein interaction, and a C-terminal region rich in tyrosine and serine residues (3-6, 10, 23, 27, 30). Tyrosine phosphorylation modulates interactions with several SH2-containing signaling molecules such as RasGAP, Nck, and the Xlinked lymphoproliferative syndrome gene product SH2D1A (3,24,26,33,43,48,49,52). Dok1 has emerged as a key negative regulator downstream of several receptor and nonreceptor tyrosine kinase cascades. Dok1 down-regulates cell proliferation and lymphocyte signaling, inhibits mitogen-activated protein (MAP) kinase activity, and mediates activin-induced apoptosis (18,31,45,(51)(52)(53).Dok1 is altered and down-regulated in chronic lymphocytic leukemia and can suppress cell transformation and leukemia (7,22,32,41,54). However, it appears not to play a major role in familial chronic lymphocytic leukemia cases (38). Knowing its tumor-suppressive activity, it is likely that genetic alterations or low expression of Dok1 and its related member Dok2 may be involved in a variety of malig...

Ksr has been genetically defined as a component of the Ras/MAP kinase pathway, but its role has been unclear. New studies now provide evidence that Ksr is important for signal transmission within the MAP kinase module, where it apparently acts as a location-regulated scaffold connecting MEK to Raf.