ERK1/2 Achieves Sustained Activation by Stimulating MAPK Phosphatase-1 Degradation via the Ubiquitin-Proteasome Pathway

Abstract

Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogenactivated protein kinase phosphatase 1 (MKP-1) protein levels in time-and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb ( Members of the family of mitogen-activated protein kinase (MAPK) 1 proteins are vital intracellular signaling components that become phosphorylated and activated in response to a wide diversity of extracellular stimuli, including growth factors, cytokines, and environmental stresses (reviewed in Refs. 1-5). MAPKs are activated through a three-kinase module composed of a MAPK, a MAPK kinase (MKK), and a MKK kinase (MKKK). These MAPK modules are connected to cell surface receptors and activated via interaction with a family of small GTPases and MKKK kinases. Activated MAPKs phosphorylate many substrates, including cytoskeletal proteins, other kinases, phosphatases, enzymes, and transcription factors, thereby orchestrating several cellular alterations including proliferation, differentiation, survival, and apoptosis. The duration and strength of MAPK activation also affects these biological outcomes. Three major MAPK subfamilies have been extensively studied, i.e. the extracellular signal-regulated kinases (ERK1/2), the c-Jun N-terminal kinases (JNKs), and the p38 kinases. Activation of a particular MAPK signal must be controlled with high specificity and efficiency to achieve precise physiological regulation. The recent discovery of specific docking sites among the members of the MAPK cascades provide a mechanism that explains how specific and efficient signaling is established. For instance, a cluster of positively charged amino acids followed by an LXL motif called the D domain (or the kinase interaction motif, KIM) has been identified in MKKs, MAPK phosphatases (MKPs), and several MAPK substrates (6 -8). The D domain binds specifically to an acidic domain (common docking domain) within a docking groove of MAPKs (6 -8). Another docking site found in many ERK substrates is called the DEF motif (docking site for ERK, FXFP) (8, 9). These docking interactions facilitate phosphorylation of substrates by MAPKs on specific Ser or Thr residues followed by a Pro residue ((S/T)P sites).The small GTPase, MKKK, and MKK in the ERK pathway are known to be Ras, Raf, and MKK1/2, respectively (1-5). Activation of ERK1/2 requires a dual-phosphorylation by MKK1/2 on the Thr and Tyr residues of TEY sites within the activation loop, wherea...

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