Isolation of two members of the rat MAP kinase kinase gene family

Mitogen-activated protein (MAP) kinase kinase (MAPKK) is a recently characterized activator of MAP kinase (MAPK), and is considered to be regulated by a protooncogene product c-Raf-1. It is, however, unclear whether the signals originating from c-Raf-1 utilize this phosphorylation cascade to lead to oncogenesis. To clarify this point, we isolated rat MAPKK cDNAs, and identified two distinct cDNAs encoding MAPKK and a highly related kinase, both with molecular weights of 5 kDa (MEK1 and MEK2). Genomic Southern blot analyses suggested that MAPKK. may form a large gene family.     

Nck-dependent activation of extracellular signal-regulated kinase-1 and regulation of cell survival during endoplasmic reticulum stress

In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1alpha-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival.     

Involvement of 3-phosphoinositide-dependent protein kinase-1 in the MEK/MAPK signal transduction pathway

The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway.     

Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1.

The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (deltaRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER) (N-BxB-ER cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 4-hydroxytamoxifen. Addition of 4-hydroxytamoxifen to N-BxB-ER cells arrested by density or serum starvation causes reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G2/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G1/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27Kip1 cyclin-dependent kinase inhibitor under all culture conditions tested.     

Prolactin induces chitotriosidase expression in human macrophages through PTK,PI3‐K,and MAPK pathways

We previously reported that prolactin (PRL) induces chitotriosidase (CHIT‐1) mRNA expression in human macrophages. In this investigation we determined the signaling pathways involved in CHIT‐1 induction in response to PRL. The CHIT‐1 induction PRL‐mediated was reduced by wortmannin and LY‐294002, inhibitors of phosphatidylinositol 3‐kinase (PI3‐K) and by genistein an inhibitor of protein tyrosine kinase (PTK). Pre‐treatment of macrophages with SB203580, a specific inhibitor of the mitogen‐activated kinases (MAPK) p38, or with U0126, an inhibitor of MAPK p44/42, prevented both basal and exogenous PRL‐mediated CHIT‐1 expression. No significant effects on CHIT‐1 induction PRL‐mediated were observed with a protein kinase C inhibitor (PKC), rottlerin, or with an Src inhibitor, PP2, or with JAK2 inhibitor, AG490. In addition, PRL induced a phosphorylation of AKT that was prevented both by the two MAPK inhibitors SB203580 and U0126 and by the PI3‐K inhibitors wortmannin and LY‐294002. In conclusion, our results indicate that PRL up‐regulated CHIT‐1 expression via PTK, PI3‐K, MAPK, and signaling transduction components. J. Cell. Biochem. 107: 881–889, 2009. © 2009 Wiley‐Liss, Inc.     

The kinase activity of kinase suppressor of Ras1 (KSR1) is independent of bound MEK

Kinase Suppressor of Ras1 (KSR1) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf-1, and pharmacologic inactivation of KSR1 may serve as a treatment for Rasdriven malignancies such as pancreatic cancer (Xing, H. R., Cordon-Cardo, C., Deng, X., Tong, W., Campodonico, L., Fuks, Z., and Kolesnick, R. (2003) Nat. Med. 9, 1266-1268). Although some studies demonstrated a requirement for KSR1 kinase activity for its action, others suggested KSR1 acts primarily as a scaffold facilitating assembly of the c-Raf-1/MEK module. We recently established a two-stage in vitro reconstitution assay to measure KSR1 kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). In this assay, KSR1, immunopurified to apparent homogeneity, never comes in contact with recombinant kinases other than c-Raf-1. In the first assay stage, activated KSR1 is incubated with recombinant c-Raf-1 and ATP. In the second stage, activated c-Raf-1 is separated from KSR1, and incubated with unactivated MEK1, unactivated MAPK, Elk-1, and ATP. Elk-1 phosphorylation serves as a specific readout for MAPK activation. However, because KSR1 constitutively associates with MEK1 and this interaction appears critical for KSR1 scaffolding function, it has been argued that the kinase activity detected is an artifact of KSR1-bound MEK1. To address these concerns, we depleted as much as 90% of KSR1-bound MEK1 by high salt washing without altering KSR1 kinase activity. Further, a complete inactivation of KSR1-bound MEK1 by pretreating with the MEK inhibitor PD 98059 prior to the first assay stage did not alter KSR1 kinase activity. In addition, the omission of exogenous recombinant GST-MEK1 from the reaction mixture during the second assay stage abolished Elk-1 phosphorylation confirming KSR1-bound MEK1 does not support MAPK activation in our in vitro assay. Moreover, a kinase-inactive mutant, FLAG-Ki-KSR1(D683A/D700A), which efficiently interacts with endogenous MEK1, lacks kinase activity. These results collectively support our contention that the kinase activity of KSR1 is an intrinsic property of this protein independent of KSR1-bound endogenous MEK.