Feedback Regulation of Raf-1 and Mitogen-activated Protein Kinase (MAP) Kinase Kinases 1 and 2 by MAP Kinase Phosphatase-1 (MKP-1)

Abstract

Inactivation of growth factor-regulated mitogen-activated protein (MAP) kinases (ERK1 and ERK2) has been proposed to occur in part through dephosphorylation by the dual specificity MAP kinase phosphatase-1 (MKP-1), an immediate early gene that is induced by mitogenic signaling. In this study, we examined the effect of MKP-1 on signaling components upstream of ERK1 and ERK2. Coexpression of MKK1 or MKK2 with MKP-1 resulted in 7-10-fold activation of mitogen-activated protein kinase kinase (MKK), which required the presence of regulatory serine phosphorylation sites. Endogenous MKK1 and MKK2 were also activated upon MKP-1 expression. Raf-1, a direct regulator of MKK1 and MKK2, was activated under these conditions, and a synergistic activation of MKK was observed upon coexpression of Raf-1 and MKP-1. This effect did not appear to involve synthesis of autocrine growth factors or the inhibition of basal extracellular signal-regulated kinase (ERK) activity but was inhibited by a dominant negative Ras mutant, indicating that MKP-1 enhances Ras-dependent activation of Raf-1 in a cell autonomous manner. This study demonstrates positive feedback regulation of Raf-1 and MKK by the MKP-1 immediate early gene and a potential mechanism for activating Raf-1/MKK signaling pathways alternative to those involving ERK. The mitogen-activated protein (MAP)1 kinase cascade has emerged as a key signaling pathway regulating factor-dependent cell growth and differentiation through intracellular phosphorylation (1, 2). Growth factor regulation of this pathway involves the phosphorylation and activation of MAP kinases, ERK1 and ERK2, by MAP kinase kinases, MKK1 and MKK2 (3-6). ERKs 1 and 2 phosphorylate various targets including upstream and downstream protein kinases, cell surface receptors, and nuclear transcription factors (1, 7). MKKs 1 and 2, in turn, can be activated through phosphorylation by members of the Raf protein kinase family, including Raf-1, which is ubiquitously expressed (8, 9). Receptor-dependent activation of Raf-1 involves its interaction with p21 Ras, through a mechanism that is not completely defined, but appears to involve Raf-1 dimerization and phosphorylation by heterologous protein kinases (10 -13).The product of the immediate early gene, MAP kinase phosphatase (MKP-1), is able to dephosphorylate phosphoserine/ threonine as well as phosphotyrosine residues, and shows selectivity for ERKs 1 and 2 in vitro, with lower activity toward other MAP kinases such as JNK and p38 MAP kinase (14, 15). MKP-1 inactivates ERK following growth factor stimulation in intact cells and also suppresses signaling downstream of ERK at the level of gene transcription and proliferation (15-17), most likely through its inhibitory effects on MAP kinase. Interestingly, MKP-1 is transcriptionally induced by stress-regulated pathways such as those occurring in response to UV treatment or activation of stress-regulated protein kinases (18,19), suggesting that MKP-1 may function as a mediator of cross-regulatory pathways between mammalian prot...

Age-dependent Decline in Mitogenic Stimulation of Hepatocytes

“…3), suggesting that MKP-1 is not regulating the age-related decrease in ERK-MAP kinase activity. Further support for the lack of a role for MKP-1 in regulating the decrease in ERK-MAP kinase comes from the work of Shapiro and Ahn (38). They reported that MKP-1 has a positive effect on Raf kinase activity independent of the basal inhibition of ERK-MAP kinase activity.…”

A different function for a critical tryptophan in c-Raf and Hck

“…The Ras to Raf to MEK to MAPK pathway has been thought to be a simple linear pathway. Recently, COT1 has been shown to activate MEK (Hagemann et al, 1999), TC21 has been shown to activate Raf (Rosario et al, 1999), MKP-1 regulates c-Raf and MAPK (Shapiro and Ahn, 1998). Clearly there are`branches' in the pathway and it may be that other molecules may also participate in the MAPK cascade in ways that have not been observed to date.…”

MEKK1 plays a critical role in activating the transcription factor C/EBP-β-dependent gene expression in response to IFN-γ

“…Catalytically inactive dominant negative (DN) mutants of ERK1 (K71R) and ERK2 (K52R) cloned in pCEP4 vector (17) were provided by M. H. Cobb (University of Texas Southwestern Medical Center, Dallas). A constitutively active (CA) MEK1 and DN-MEK1 cloned in pCMV were described (18,19). Wild type, CA-MEKK1 (⌬367), and DN-MEKK1 (⌬367…”