Microtubule Integrity Regulates Pak Leading to Ras-independent Activation of Raf-1

Abstract

Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser 338 . Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/ Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser 338 to Ala or Tyr 340 -Tyr 341 to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4␤-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of 338 SSYY 341 ; 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser 338 , an event crucial for Raf activation.The proto-oncogene raf-1, first identified as a cellular counterpart of the oncogene v-raf, encodes a serine/threonine protein kinase. Raf-1 is ubiquitously expressed and plays an important role in cell proliferation and differentiation (1). The mechanism by which Raf-1 is activated by growth factors is still incompletely understood, although it is known to be preassembled as a complex with 14-3-3 and heat shock proteins 90/50 (2-6). It involves multiple steps including Ras-GTP binding, membrane recruitment, and phosphorylation.Raf-1 consists of an amino-terminal regulatory domain and a carboxyl-terminal kinase domain. The amino-terminal moiety of Raf-1 exerts an inhibitory effect on the catalytic activity, since amino-terminal truncations lead to progressive increases in its transforming ability (7,8). The amino-terminal regulatory region of Raf-1 contains a Ras binding domain (RBD) 1 and a cysteine-rich zinc finger domain (CRD), both of which participate in binding to Ras. The first interaction engages Raf RBD ranging from ...

AMP-activated Protein Kinase Is Required for the Lipid-lowering Effect of Metformin in Insulin-resistant Human HepG2 Cells

“…Western Blot Analysis-Western blot analysis was carried out as described previously (30,31). In brief, HepG2 cells were lysed in buffer (20 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 2 g/ml leupeptin, and 1 g/ml pepstatin).…”

“…After fresh serum-free medium was added, cells were treated with metformin (0.5-2 mM) for the indicated times. Preliminary studies revealed that within 48 h of transfection with control Ad-GFP, 80 -90% of HepG2 cells expressed green fluorescent protein.Western Blot Analysis-Western blot analysis was carried out as described previously (30,31). In brief, HepG2 cells were lysed in buffer (20 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 2 g/ml leupeptin, and 1 g/ml pepstatin).…”

B-Raf and Raf-1 Are Regulated by Distinct Autoregulatory Mechanisms

“…We then tested whether the phosphorylation of Ser 445 could be increased by expression of constitutively active Ras or constitutively active PAK1 because Ras stimulates B-Raf activity and because PAK1 phosphorylates Raf-1 at the analogous site (Ser 338 ) (28,36,38,42). The B-Raf N Terminus Contains an Autoinhibitory DomainRaf-1 contains an autoinhibitory domain that minimally encompasses residues 1-147 and that includes the entire RBD and part of the CRD.…”

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis

“…In the present study, the inability of mda-5 to induce apoptosis in CREF-raf cells even after blocking K-ras activity by Ad.Kras(AS) approaches indicates a Ras-independent Raf activation, which was also documented in previous studies. 34,36 However, the susceptibility of CREF-raf to mda-5-induced killing after MEK1/2 inhibition by PD98059 further confirms the involvement of Raf/ MEK/ERK in mda-5-mediated apoptosis. Studies are in progress to analyze the association of Ras/Raf/MEK/ERK pathway with different aspects of the apoptotic machinery involved in Ad.mda-5-induced apoptosis such as bcl-2 family members, as they have been related to Ras/Raf/MEK/ERKmodulated cell survival.…”