Oncogenic Ras abrogates MEK SUMOylation that suppresses the ERK pathway and cell transformation.

The ERK (extracellular signal-regulated kinase) MAPK (mitogen-activated protein kinase) cascade (Raf-MEK-ERK) mediates mitogenic signalling, and is frequently hyperactivated by Ras oncogenes in human cancer. The entire range of activities of multifunctional Ras in carcinogenesis remains elusive. Here we report that the ERK pathway is downregulated by MEK (MAPK-ERK kinase) SUMOylation, which is inhibited by oncogenic Ras. MEK SUMOylation blocked ERK activation by disrupting the specific docking interaction between MEK and ERK. Expression of un-SUMOylatable MEK enhanced ERK activation, cell differentiation, proliferation and malignant transformation by oncogenic ErbB2 or Raf, but not by active Ras. Interestingly, MEK SUMOylation was abrogated in cancer cells harbouring Ras mutations. Oncogenic Ras inhibits MEK SUMOylation by impairing the function of the MEKK1 MAPKKK as a SUMO-E3 ligase specific for MEK. Furthermore, forced enhancement of MEK SUMOylation suppressed Ras-induced cell transformation. Thus, oncogenic Ras efficiently activates the ERK pathway both by activating Raf and by inhibiting MEK SUMOylation, thereby inducing carcinogenesis.

Stimulus-specific distinctions in spatial and temporal dynamics of stress-activated protein kinase kinase kinases revealed by a fluorescence resonance energy transfer biosensor.

The stress-activated protein kinases (SAPKs), namely, p38 and JNK, are members of the mitogen-activated protein kinase family and are important determinants of cell fate when cells are exposed to environmental stresses such as UV and osmostress. SAPKs are activated by SAPK kinases (SAP2Ks), which are in turn activated by various SAP2K kinases (SAP3Ks). Because conventional methods, such as immunoblotting using phospho-specific antibodies, measure the average activity of SAP3Ks in a cell population, the intracellular dynamics of SAP3K activity are largely unknown. Here, we developed a reporter of SAP3K activity toward the MKK6 SAP2K, based on fluorescence resonance energy transfer, that can uncover the dynamic behavior of SAP3K activation in cells. Using this reporter, we demonstrated that SAP3K activation occurs either synchronously or asynchronously among a cell population and in different cellular compartments in single cells, depending on the type of stress applied. In particular, SAP3Ks are activated by epidermal growth factor and osmostress on the plasma membrane, by anisomycin and UV in the cytoplasm, and by etoposide in the nucleus. These observations revealed previously unknown heterogeneity in SAPK responses and supplied answers to the question of the cellular location in which various stresses induce stimulus-specific SAPK responses.

Workshop cluster 1, a gammadelta T cell specific receptor is phosphorylated and down regulated by activation induced Src family kinase activity.

Workshop cluster 1(+) gamma delta (WC1(+)gammadelta) T cells have been shown to play important roles in the immune response to infections. WC1 is a transmembrane glycoprotein, uniquely expressed on the surface of gammadelta T cells of ruminants and pigs. A role for WC1 in inducing a reversible growth arrest of gammadelta T cells has been previously demonstrated. WC1-induced growth inhibition has been shown to be overcome following gammadelta T cell activation with Concanavalin A (Con A). However, molecular mechanism(s) by which WC1 signalling might be modulated following activation have not been elucidated. In this paper we show that Con A activation of bovine lymphocytes induces the tyrosine phosphorylation of WC1 in a Src-family kinase-dependent manner. Src family kinases also phosphorylated WC1 in a COS-7 co-transfection system. Furthermore, a glutathione-S-transferase (GST)-WC1 cytoplasmic domain fusion protein was directly phosphorylated by recombinant Lck (rLck) in vitro. The Y(1303) of WC1 was identified by mutational analysis as the only one of the five WC1 tyrosine residues to be critical for Src family phosphorylation. The importance of activation-induced Src family activity for WC1 function was investigated with the Src-family specific inhibitor PP2. These studies show that the surface levels of WC1 are down regulated in a Src-family-dependent manner following activation of bovine lymphocytes. Down regulation of surface WC1 was accompanied by a Src-family-dependent accumulation of intracellular WC1. These data show that WC1 is modulated by activation-induced tyrosine phosphorylation thus providing a new insight into the signalling mechanisms by which WC1 and gammadelta T cell activation are regulated in this important and unique cell population.