S338 Phosphorylation of Raf-1 Is Independent of Phosphatidylinositol 3-Kinase and Pak3

Chiloeches, Antonio; Mason, Clive; Marais, Richard

doi:10.1128/mcb.21.7.2423-2434.2001

Cited by 66 publications

(63 citation statements)

References 61 publications

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“…In accordance with findings by others (31), our data suggest that Rac-1 additionally is involved in the pathway from Ras to the cyclin D1 promoter via the Ras Ͼ Raf Ͼ ERK cascade, presumably by activating p65 PAK-1 (32). The recent suggestion that PAK-3 could stimulate Raf-1 activity by directly phosphorylating Ser-338 through a Ras-phosphatidylinositol 3-kinase/Cdc42-dependent pathway has attracted much attention (48). However, in HC11 cells, this pathway seems to be of minor importance, as the PI 3-kinase inhibitors wortmannin and LY 294002 did not interfere with cyclin D1 induction by Ras (not shown).…”

Section: Discussionsupporting

confidence: 77%

Protein Kinase C Isoforms Involved in the Transcriptional Activation of Cyclin D1 by Transforming Ha-Ras

Kampfer

Windegger²,

Hochholdinger³

et al. 2001

Journal of Biological Chemistry

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Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors which interact with distinct elements of the cyclin D1 promoter. The present investigations revealed that cyclin D1 induction by transforming Ha-Ras is MEKand Rac-dependent and requires the PKC isotypes ⑀, , and , but not cPKC-␣. This conclusion is based on observations indicating that cyclin D1 induction by transforming Ha-Ras was depressed in a dose-dependent manner by PD98059, a selective inhibitor of the mitogenactivated kinase kinase MEK-1, demonstrating that HaRas employs extracellular signal-regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes ⑀ and , but not are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-⑀ or aPKC-inhibit ERK activation by constitutively active Raf-1. Phosphorylation within the TEY motif and subsequent activation of ERKs by constitutively active MEK-1 was significantly inhibited by DN aPKC-, indicating that aPKC-functions downstream of MEK-1 in the pathway leading to cyclin D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-⑀, suggesting another position for this kinase within the cascade investigated. Transformation by oncogenic Ras requires activation of several Ras effector pathways which may be PKC-dependent and converge on the cyclin D1 promoter. Therefore, we investigated a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulation of cyclin D1. We have been able to reveal that cyclin D1 induction by oncogenic Ha-Ras is Rac-dependent and requires the PKC isotypes ⑀, , and , but not cPKC-␣. Evidence is presented that aPKC-acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes ⑀ and act downstream of Rac and are required for the activation of ERKs.Transformation by oncogenic Ras requires activation of several Ras effectors which, in turn, stimulate different signaling pathways. These Ras effectors include Raf, Ral-GDS, PI 3-kinase, and Rac-1 (1). Activation of Rac-1 by oncogenic Ras is mediated by PI 3-kinase 1 -dependent (2, 3) and PI 3-kinaseindependent mechanisms (2). These Ras effector pathways may converge on the cyclin D1 promoter (1, 4). Evidence for a positive regulation of the cyclin D1 promoter by Ras has been presented (4 -7).The Ras-Raf-ERK cascade has been described as the dominant pathway by which Ras transmits signals to the cyclin D1 promoter (4, 8 -10), although in nontransformed cells the situation may be different (1). One effect of the stimulation of ERK1 by Ras seems to be the activation of Ets-2, since expression of plasmids encoding DN Ets (Ets-LacZ) antagonized ERKdependent activation of the cyclin D1 promoter (4) and mutation of an Ets-binding site (termed EtsB (6)) strongly reduced basal and also Ras-induced activation of cyclin D1.Another Ras effector, Rac-1, has also been...

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Section: Discussionsupporting

confidence: 77%

Protein Kinase C Isoforms Involved in the Transcriptional Activation of Cyclin D1 by Transforming Ha-Ras

Kampfer

Windegger²,

Hochholdinger³

et al. 2001

Journal of Biological Chemistry

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“…Therefore, the requirement of raft localization for full Raf-1 activity is coupled to Ser-338 phosphorylation, extending previous studies showing that the membranelocalized Ser-338 kinase was required for Raf-1 activation by oncogenic Ras (29). A candidate kinase, PAK, has been proposed (49 -51); however, its role has been challenged (52).…”

Section: Ser-338 Phosphorylation Of Raf-1 Requires Targeting To Raftsupporting

confidence: 52%

The Requirement of Specific Membrane Domains for Raf-1 Phosphorylation and Activation

Carey

Watson

Pessin

et al. 2003

Journal of Biological Chemistry

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Activation of Raf-1 by Ras requires recruitment to the membrane as well as additional phosphorylations, including phosphorylation at serine 338 (Ser-338) and tyrosine 341 (Tyr-341). In this study we show that Tyr-341 participates in the recruitment of Raf-1 to specialized membrane domains called "rafts," which are required for Raf-1 to be phosphorylated on Ser-338. Raf-1 is also thought to be recruited to the small G protein Rap1 upon GTP loading of Rap1. However, this does not result in Raf-1 activation. We propose that this is because Raf-1 is not phosphorylated on Tyr-341 upon recruitment to Rap1. Redirecting Rap1 to Ras-containing membranes or mimicking Tyr-341 phosphorylation of Raf-1 by mutation converts Rap1 into an activator of Raf-1. In contrast to Raf-1, B-Raf is activated by Rap1. We suggest that this is because B-Raf activation is independent of tyrosine phosphorylation. Moreover, mutants that render B-Raf dependent on tyrosine phosphorylation are no longer activated by Rap1.The mitogen-activated protein (MAP) 1 kinase family regulates diverse physiological processes including cell growth, differentiation, and death. Activation of one of these MAP kinases (the extracellular signal-regulated kinase or ERK) is initiated by the recruitment of the MAP kinase kinase kinase Raf-1 to the small G protein Ras, a resident plasma membrane protein.The Ras family consists of three members (Ha-Ras, Ki-Ras, and N-Ras) (1) that display overlapping but distinct patterns of expression and function (2). Ras is tethered to the membrane via a carboxyl CAAX motif containing a cysteine followed by two aliphatic amino acids (A) and a carboxyl-terminal amino acid (X) that directs the attachment of a farnesyl moiety (3, 4). In addition to farnesylation, a second signal assists in correct membrane targeting. For Ha-Ras and N-Ras, this second signal is a palmitoyl moiety that is introduced on a neighboring cysteine. In Ki-Ras, this second site is a polybasic domain.

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“…However, these mutations alone do not activate Raf-1 kinase, implying that phosphorylation of serine 338 may contribute to, but does not determine Raf-1 activation. It has also been suggested that S338 phosphorylation occurs independently of PI3K-dependent PAK activity and hypothesised that other kinases may target this residue (Chiloeches et al, 2001). Both serines are conserved in B-Raf but the counterpart of serine 338 appears to be constitutively phosphorylated (Mason et al, 1999).…”

Section: Pak1 Phosphorylation Of Raf-1 On Serine 338mentioning

confidence: 99%

PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway

2002

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The serine/threonine kinase Raf-1 acts downstream of Ras in the MAPK pathway leading to ERK activation in response to mitogens. Raf-1 has oncogenic potential, but is normally controlled by a complex interplay of inhibitory and activating mechanisms. Although Raf-1 is phosphorylated in unstimulated cells, mitogens cause its membrane recruitment by Ras and subsequent phosphorylation on additional sites. Some of these events modulate Raf-1 kinase activity while others determine interactions with other proteins. These changes regulate the ability of Raf-1 to phosphorylate its downstream targets MEK1 and MEK2. Rho family small G proteins act synergistically with Raf-1 to stimulate the ERK pathway by a cross-cascade mechanism that enhances MEK phosphorylation by Raf-1. Here we show that both Raf-1 and MEK1 are phosphorylated by PAK1 and that mutations at PAK1 phosphorylation sites in either protein prevent cross-cascade activation. In contrast, MEK1 activation by constitutively-active Raf-1 is refractory to mutations at PAK1 phosphorylation sites. Phosphorylation of MEK1 on serine 298 does not appear to regulate the interaction between Raf-1 and MEK1, but rather the ability of Raf-1 to phosphorylate MEK1 with which it is complexed in vivo. Our ®ndings indicate that PAK1 primes MEK1 for activation by Raf-1 and imply another level of regulation in the ERK cascade.

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S338 Phosphorylation of Raf-1 Is Independent of Phosphatidylinositol 3-Kinase and Pak3

Cited by 66 publications

References 61 publications

Protein Kinase C Isoforms Involved in the Transcriptional Activation of Cyclin D1 by Transforming Ha-Ras

Protein Kinase C Isoforms Involved in the Transcriptional Activation of Cyclin D1 by Transforming Ha-Ras

The Requirement of Specific Membrane Domains for Raf-1 Phosphorylation and Activation

PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway

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