Isolation of a new protein factor required for activation of Raf-1 by Ha-Ras: partial purification from rat brain cytosols

Mizutani, Shin; Koide, Hiroshi; Kaziro, Yoshito

doi:10.1038/sj.onc.1201806

Cited by 7 publications

(20 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the extent of activation appeared to be very slight, compared to that achieved in viva, and also appeared to require the continued presence of Ras-GTP. We infer that this reflects an intermediate step in the conversion of Raf to a stably activated, Ras-free form, a view supported by other work (Mizutani et al, 1998). As indicated earlier, such stable activation most often reflects the introduction of a post-translational modification, usually phosphorylation.…”

Section: Conversion Of Craf-1 To a Stably Active Statesupporting

confidence: 52%

Ras Activation of the Raf Kinase: Tyrosine Kinase Recruitment of the MAP Kinase Cascade

Avruch¹

2001

Recent Progress in Hormone Research

347

234

View full text Add to dashboard Cite

A continuing focus of our work has been an effort to understand the signal transduction pathways through which insulin achieves its cellular actions. In the mid-1970s, we and others observed that insulin promoted an increase in Ser/Thr phosphorylation of a subset of cellular proteins. This finding was unanticipated, inasmuch as nearly all of the actions of insulin then known appeared to result from protein dephosphorylation.In fact, nearly 15 years elapsed before any physiologic response to insulin attributable to stimulated (Ser/Thr) phosphorylation was established. Nevertheless, based on the hypothesis that insulin-stimulated Sernhr phosphorylation reflected the activation of protein (Sermhr) kinases downstream of the insulin receptor, we sought to detect and purify these putative, insulin-responsive protein (SerDhr) kinases. Our effort was based on the presumption that an understanding of the mechanism for their activation would provide an entry into the biochemical reactions through which the insulin receptor activated its downstream effecters. To a degree that, in retrospect, is surprising, this goal was accomplished, much in the way originally envisioned. It is now well known that receptor tyrosine kinases (RTKs) recruit a large network of protein (SerIThr) kinases to execute their cellular programs.The first of these insulin-activated protein kinase networks to be fully elucidated was the Ras-Raf-mitogen-activated protein kinase (MAPK) cascade. This pathway is a central effector of cellular differentiation in development; moreover, its inappropriate and continuous activation provides a potent promitogenic force and is a very common occurrence in human cancers. Conversely, this pathway contributes minimally, if at all, to insulin's program of metabolic regulation. Nevertheless, the importance ofthe Raa-MAPK pathway in metazoan biology and human malignancies has impelled us to an ongoing analysis of the functions and regulation of Ras and Raf. This chapter will summarize briefly the way in which work from this and other laboratories on insulin signaling led to the discovery of the mammalian MAP kinase cascade and, in turn, to the identification of unique role of the Raf kinases in RTK activation of this protein (Ser/Thr) kinase cascade. We will then review in more detail current understanding of the biochemical mechanism through which the Ras proto-oncogene, in collaboration with the 14-3-3 protein and other protein kinases, initiates activation of the Raf kinase. 127

show abstract

Section: Conversion Of Craf-1 To a Stably Active Statesupporting

confidence: 52%

Ras Activation of the Raf Kinase: Tyrosine Kinase Recruitment of the MAP Kinase Cascade

Avruch¹

2001

Recent Progress in Hormone Research

347

234

View full text Add to dashboard Cite

show abstract

“…We and others consistently observed that the CRD-Ras interaction is independent of the guanine nucleotide configuration of Ras and requires both the C-terminal posttranslational modifications and the intact activator region of Ras (15,17,21,38), a finding confirmed by the present study. This is consistent with the observation that activator region mutants of Ras lack the ability to activate Raf-1 while retaining the ability to associate with RBD (43) as well as with the results of studies using in vitro cell-free systems, which found an absolute dependence of the Raf activation on the C-terminal posttranslational modifications of Ras (32,41,50) even though another cellular factor seems to be required for achieving Ras-dependent activation of Raf-1 and B-Raf (11,26,41). In contrast, studies by other groups found that the CRD-Ras interaction exhibits a GTP dependence (3,12), is independent of the posttranslational modifications of Ras (9), and is abolished by mutations in the switch II region of Ras (12).…”

Section: Discussionmentioning

confidence: 99%

“…This observation was interpreted to indicate that the CRD-CR3 interaction affects the availability of CRD for interaction with Ras. Studies using an in vitro cell-free system for Ras-dependent Raf activation suggested the presence of an unknown factor essential for the activation (11,26,41). This factor might also be a candidate for competition with Ras and/or Rap1A.…”

Section: Discussionmentioning

confidence: 99%

The Strength of Interaction at the Raf Cysteine-Rich Domain Is a Critical Determinant of Response of Raf to Ras Family Small GTPases

Okada

Jin

et al. 1999

Molecular and Cellular Biology

View full text Add to dashboard Cite

To be fully activated at the plasma membrane, Raf-1 must establish two distinct modes of interactions with Ras, one through its Ras-binding domain and the other through its cysteine-rich domain (CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and even antagonizes Ras-dependent activation of Raf-1. We proposed previously that this property of Rap1A may be attributable to its greatly enhanced interaction with Raf-1 CRD compared to Ras. On the other hand, B-Raf, another Raf family member, is activatable by both Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Raf CRD did not exhibit the enhanced interaction with Rap1A, suggesting that the strength of interaction at CRDs may account for the differential action of Rap1A on Raf-1 and B-Raf. The importance of the interaction at the CRD is further supported by a domain-shuffling experiment between Raf-1 and B-Raf, which clearly indicated that the nature of CRD determines the specificity of response to Rap1A: Raf-1, whose CRD is replaced by B-Raf CRD, became activatable by Rap1A, whereas B-Raf, whose CRD is replaced by Raf-1 CRD, lost its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is selectively enhanced was isolated and found to possess the double mutation K252E/M278T. B-Raf carrying this mutation was not activated by Rap1A but retained its response to Ras. These results indicate that the strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family small GTPases.Raf-1 is a serine/threonine kinase that plays a pivotal role in conveying a signal from receptor tyrosine kinases and Ras to the mitogen-activated protein kinase (MAPK) cascade. Although it is well established that Raf-1 interacts directly with the GTP-bound active form of Ras, the precise mechanism by which Raf-1 is activated by interaction with Ras is not known (for reviews, see references 1, 4, and 27). In addition to Raf-1, which is found in a variety of mammalian tissues, two close homologues, B-Raf and A-Raf, have been identified. These exhibit more limited tissue distribution: B-Raf expression is confined to the brain and testis, and A-Raf is expressed most abundantly in the ovary and epididymis (42). All Raf kinases possess three distinct regions designated conserved region 1 (CR1), CR2, and CR3 (for a review, see reference 10). The N-terminal regulatory domain contains CR1 and CR2, while the C-terminal CR3 corresponds to the protein kinase catalytic domain. CR1 consists of two distinct structural modules called the Ras-binding domain (RBD; residues 51 to 131) and the cysteine-rich domain (CRD; residues 139 to 184). RBD has a ubiquitin superfold (30) and constitutes a principal interface for GTP-dependent interaction with Ras and Rap1A (5, 46, 49). CRD consists of two zinc finger motifs resembling the C1 domain of protein kinase C, a phorbol ester-binding site (28). Constitutive activation of Raf-1 by N-terminal truncations indicated that the N-terminal reg...

show abstract

“…Then cells were harvested and lysed with lysis buffer (10 mM HEPES/NaOH (pH 7.4), 10 mM MgCl 2 , 100 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 mM NaF, 25 mM ␤-glycerophosphate, 4 g/ml aprotinin, 10 g/ml leupeptin, 1% Triton X-100, 10% glycerol). In all experiments, kinase activity of RafFH was determined using His-MEK and GST-kdMAPK, kind gifts from E. Nishida (Kyoto University), as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

“…However, since direct interaction of Ras with Raf-1 is insufficient for Raf-1 activation (5, 7), an additional molecule(s) has been expected to be involved in this activation. In fact, using a cell-free system, we have found a Ras-dependent Raf-1 activator in the cytosolic fraction (8).…”

mentioning

confidence: 99%

Formation of the Ras Dimer Is Essential for Raf-1 Activation

Inouye

Mizutani

Koide

et al. 2000

Journal of Biological Chemistry

Self Cite

146

133

View full text Add to dashboard Cite

Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cellfree system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation.Ras GTPases (Ha-, Ki-, and N-Ras) are the key proteins in eukaryotic signal transduction directed toward cellular proliferation and differentiation (1-3). The biological activity of Ras is controlled by a regulated GDP/GTP cycle. Guanine-nucleotide exchange factors (Ras-GRF1/2, mSos1/2) induce dissociation of GDP from Ras⅐GDP to form an active, GTP-bound form of Ras. On the other hand, GTPase-activating proteins (p120GAP, NF1) accelerate the intrinsic GTP hydrolytic activity of Ras to promote the formation of an inactive, GDP-bound form of Ras. Upon binding of GTP, Ras alters its conformation to interact with multiple downstream effectors. One of the well characterized effectors is a serine/threonine kinase Raf-1 (4, 5), which induces activation of a dual specificity kinase MEK.1 Activated MEK in turn activates a serine/threonine kinase ERK, which phosphorylates a variety of proteins including protein kinases, transcription factors, and cytoskeletal proteins (6).Although it has been demonstrated that Ras binds to Raf-1 directly, the precise mechanism of Raf-1 activation by Ras is not fully understood. According to the current model (5), when Ras is activated, the effector region of Ras interacts with the Ras binding domain of Raf-1, which leads to the binding of cysteine-rich domain of Raf-1 to Ras. These interactions relieve the masking of the C-terminal catalytic domain of Raf-1 by the N-terminal regulatory domain and allow Raf-1 to interact with the downstream kinase, MEK. However, since direct interaction of Ras with Raf-1 is insufficient for Raf-1 activation (5, 7), an additional molecule(s) has been expected to be involved in this activation. In fact, using a cell-free system, we have found a Ras-dependent Ra...

show abstract

Isolation of a new protein factor required for activation of Raf-1 by Ha-Ras: partial purification from rat brain cytosols

Cited by 7 publications

References 38 publications

Ras Activation of the Raf Kinase: Tyrosine Kinase Recruitment of the MAP Kinase Cascade

Ras Activation of the Raf Kinase: Tyrosine Kinase Recruitment of the MAP Kinase Cascade

The Strength of Interaction at the Raf Cysteine-Rich Domain Is a Critical Determinant of Response of Raf to Ras Family Small GTPases

Formation of the Ras Dimer Is Essential for Raf-1 Activation

Contact Info

Product

Resources

About