On the mitogenic properties of Rap1b: cAMP-induced G
            <sub>1</sub>
            /S entry requires activated and phosphorylated Rap1b

Ribeiro-Neto, Fernando; Urbani, Julie; Lemee, Nicolas; Lou, Liguang; Altschuler, Daniel L.

doi:10.1073/pnas.082122499

Cited by 70 publications

(79 citation statements)

References 54 publications

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“…In support of our findings, rap1, which is inactivated by rap1GAP, is required for cAMPinduced G 1 /S transition during mitogenesis in rat thyroid cells. 19 There are other examples of GAP proteins influencing cell-cycle progression. Tuberin/TSC2, a protein with a rap1GAP homology region, inactivates rheb, a rap1-like GTP-binding protein.…”

Section: Discussionmentioning

confidence: 99%

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Rap1GAP Inhibits Tumor Growth in Oropharyngeal Squamous Cell Carcinoma

Zhang

Mitra

Henson

et al. 2006

The American Journal of Pathology

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Rap1, a growth regulatory protein that is strongly expressed in human squamous cell carcinoma (SCC), is inactivated by rap1GAP. Recent evidence in normal rat cells suggests that rap1GAP regulates proliferation. The objective of the current study was to investigate whether rap1GAP functions as a tumor suppressor in SCC. Using a pull-down assay, active GTP-bound rap1 was up-regulated in SCC compared to normal or immortalized keratinocytes. Because both rap1A and rap1B isoforms of rap1 are expressed in SCC, the rap1GAP inactivation of both rap1 isoforms was verified using cells transfected with EGFP-rap1A or EGFP-rap1B or co-transfected with FLAG-tagged rap1GAP. The results demonstrate that expression of rap1GAP in oropharyngeal SCC down-regulated active rap1, ERK activation, and proliferation. Rap1A and rap1B are ras-like proteins that are ubiquitously expressed. Rap1A, which is also referred to as smg p21 or Krev1, shares 95% homology with rap1B, differing by only nine amino acids (of a total of 184), six of which are located at the C-terminus between amino acids 171 to 184.1 Active rap1 modulates cellular functions by regulating the translocation of other proteins or by shuttling between intracellular compartments.

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Section: Discussionmentioning

confidence: 99%

“…In rat thyroid cells and subsequently in the thyroid gland of a transgenic mouse, endogenous rap1B has been shown to have a mitogenic effect. 18,19 However, little is known about the effects of inactivation of endogenous rap1 in malignant cells.…”

mentioning

confidence: 99%

Rap1GAP Inhibits Tumor Growth in Oropharyngeal Squamous Cell Carcinoma

Zhang

Mitra

Henson

et al. 2006

The American Journal of Pathology

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“…This model may explain why constitutively active Rap1 mutants are poor activators of ERKs in the absence of PKA phosphorylation and may explain why constitutively active Rap1 mutants are rarely seen in human cancers (60). In this regard, it is interesting that the mitogenic actions of constitutively active Rap1 in a model of thyroid follicular cell growth required Rap1 phosphorylation (61). In addition, endogenous levels of KSR may be limiting during overexpression of constitutively active Rap1 mutants.…”

Section: Ksr Is Required For B-raf To Bind Rap1 and Signal To Erks-mentioning

confidence: 99%

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Takahashi

Dillon

et al. 2017

Journal of Biological Chemistry

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Edited by Jeffrey E. PessinCyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1 have been proposed to mediate this activation. Using HEK293 cells as a model system, we have recently shown that both Ras and Rap1 are required for cAMP signaling to ERKs. However, cAMP-dependent Ras signaling to ERKs is transient and rapidly terminated by PKA phosphorylation of the Raf isoforms C-Raf and B-Raf. In contrast, cAMP-dependent Rap1 signaling to ERKs and Rap1 is potentiated by PKA. We show that this is due to sustained binding of B-Raf to Rap1. One of the targets of PKA is Rap1 itself, directly phosphorylating Rap1a on serine 180 and Rap1b on serine 179. We show that these phosphorylations create potential binding sites for the adaptor protein 14-3-3 that links Rap1 to the scaffold protein KSR. These results suggest that Rap1 activation of ERKs requires PKA phosphorylation and KSR binding. Because KSR and B-Raf exist as heterodimers within the cell, this binding also brings B-Raf to Rap1, allowing Rap1 to couple to ERKs through B-Raf binding to Rap1 independently of its Ras-binding domain.Hormones that are coupled to the second messenger cyclic adenosine monophosphate (cAMP) signal to multiple intracellular pathways. One of these, the MAP kinase, or ERK (extracellular signal-regulated kinase) cascade, mediates many physiological functions of cAMP in development (1), metabolism (2, 3), cognition (4 -6), and cell growth (7-12). In all cases, cAMP signaling to ERKs requires the recruitment of the MAP kinase kinase kinase Raf to one of two small G proteins, Ras or Rap1. The ability to signal to both Ras and Rap1 allows cAMP to trigger the sustained activation of ERKs, which is required for many physiological functions, including the transcription/stabilization of many transcription factors (13,14).In addition to its ability to activate both Ras and Rap1, cAMP can inhibit signaling to Ras. PKA antagonizes Ras signaling through its phosphorylation of the Raf isoform C-Raf on serine 259 (Ser-259) (15). This phosphorylation and subsequent recruitment of 14-3-3 result in the release of C-Raf from active Ras (15, 16). We have recently shown that PKA phosphorylation of the homologous site in B-Raf (Ser-365) similarly prevents the direct binding of B-Raf to Ras (16). Despite this phosphorylation of B-Raf, B-Raf is still capable of binding to Rap1. This is physiologically relevant, as Rap1/B-Raf links PKA to ERKs (17, 18). How Rap1 is able to couple to B-Raf following PKA phosphorylation of B-Raf is unknown.We and others have identified Rap1 as a target for phosphorylation by PKA (19 -21). Here, we show that Rap1 phosphorylation plays a role in the ability of B-Raf to remain bound to Rap1 following cAMP stimulation. Rap1 phosphorylation creates a novel 14-3-3 binding site that connects Rap1 to the scaffold KSR. Because KSR can exist as a dimer with B-...

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“…From this initial observation, cAMP-and cGMP-dependent kinase (PKA and PKG) were demonstrated to phosphorylate RhoA on Ser 188 (13,14), thereby joining RhoA with Rap1a and Rap1b as small GTPases regulated by carboxyl PKA phosphorylation (15,16). Although PKA phosphorylation is linked to Rap activation (17,18), evidence indicates that it negatively regulates RhoA function. RhoA phosphorylation promotes formation of RhoA⅐RhoGDI complexes (13,19) and enhances the ability of RhoGDI to extract RhoA from membranes (13,20).…”

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confidence: 99%

Serine Phosphorylation Negatively Regulates RhoA in Vivo

Ellerbroek

Wennerberg

Burridge

2003

Journal of Biological Chemistry

290

235

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Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMPdependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser 188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.

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On the mitogenic properties of Rap1b: cAMP-induced G ₁ /S entry requires activated and phosphorylated Rap1b

Cited by 70 publications

References 54 publications

Rap1GAP Inhibits Tumor Growth in Oropharyngeal Squamous Cell Carcinoma

Rap1GAP Inhibits Tumor Growth in Oropharyngeal Squamous Cell Carcinoma

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Serine Phosphorylation Negatively Regulates RhoA in Vivo

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On the mitogenic properties of Rap1b: cAMP-induced G 1 /S entry requires activated and phosphorylated Rap1b

Cited by 70 publications

References 54 publications

Rap1GAP Inhibits Tumor Growth in Oropharyngeal Squamous Cell Carcinoma

Rap1GAP Inhibits Tumor Growth in Oropharyngeal Squamous Cell Carcinoma

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Serine Phosphorylation Negatively Regulates RhoA in Vivo

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On the mitogenic properties of Rap1b: cAMP-induced G ₁ /S entry requires activated and phosphorylated Rap1b