Tara J. Dillon

Rap1 mediates sustained MAP kinase activation induced by nerve growth factor

Activation of mitogen-activated protein (MAP) kinase (also known as extracellular-signal-regulated kinase, or ERK) by growth factors can trigger either cell growth or differentiation. The intracellular signals that couple growth factors to MAP kinase may determine the different effects of growth factors: for example, transient activation of MAP kinase by epidermal growth factor stimulates proliferation of PC12 cells, whereas they differentiate in response to nerve growth factor, which acts partly by inducing a sustained activation of MAP kinase. Here we show that activation of MAP kinase by nerve growth factor involves two distinct pathways: the initial activation of MAP kinase requires the small G protein Ras, but its activation is sustained by the small G protein Rap1. Rap1 is activated by CRK adaptor proteins and the guanine-nucleotide-exchange factor C3G, and forms a stable complex with B-Raf, an activator of MAP kinase. Rap1 is required for at least two indices of neuronal differentiation by nerve growth factor: electrical excitability and the induction of neuron-specific genes. We propose that the activation of Rap1 by C3G represents a common mechanism to induce sustained activation of the MAP kinase cascade in cells that express B-Raf.

Rap1-Mediated Activation of Extracellular Signal-Regulated Kinases by Cyclic AMP Is Dependent on the Mode of Rap1 Activation

ABSTRACT Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.

PKA phosphorylation of Src mediates Rap1 activation in NGF and cAMP signaling in PC12 cells

Recent studies suggest that the tyrosine kinase Src plays an important role in the hormonal regulation of extracellular signal-regulated kinases (ERKs) via cyclic AMP (cAMP). Src has also been proposed to mediate signals downstream of nerve growth factor (NGF). Here, we report that the cAMP-dependent protein kinase A (PKA) induced the phosphorylation of Src at residue serine17 (S17) in multiple cell types including PC12, Hek293, AtT-20 and CHO cells. In PC12 cells, Src phosphorylation on S17 participates in the activation of the small G protein Rap1 by both cAMP and NGF. In these cells, Rap1 is required for cAMP/PKA signaling to ERKs and also for the sustained activation of ERKs by NGF. The activation of Rap1 by both cAMP and NGF was blocked by PP2, an inhibitor of Src family kinases, and by a Src mutant incapable of being phosphorylated by PKA (SrcS17A), consistent with the requirement of PKA phosphorylation of Src at S17 in these actions. PP2 and SrcS17A also inhibited the Rap1-dependent activation of ERKs by both agents. These results strongly indicate that PKA phosphorylation of Src at S17 is essential for cAMP and NGF signaling in PC12 cells and identify PKA as an important downstream target of NGF. PKA phosphorylation of Src may therefore be required for Rap1 activation in PC12 cells.

CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

ABSTRACT Proliferation of T cells via activation of the T-cell receptor (TCR) requires concurrent engagement of accessory costimulatory molecules to achieve full activation. The best-studied costimulatory molecule, CD28, achieves these effects, in part, by augmenting signals from the TCR to the mitogen-activated protein (MAP) kinase cascade. We show here that TCR-mediated stimulation of MAP kinase extracellular-signal-regulated kinases (ERKs) is limited by activation of the Ras antagonist Rap1. CD28 increases ERK signaling by blocking Rap1 action. CD28 inhibits Rap1 activation because it selectively stimulates an extrinsic Rap1 GTPase activity. The ability of CD28 to stimulate Rap1 GTPase activity was dependent on the tyrosine kinase Lck. Our results suggest that CD28-mediated Rap1 GTPase-activating protein activation can help explain the augmentation of ERKs during CD28 costimulation.

Ras Is Required for the Cyclic AMP-Dependent Activation of Rap1 via Epac2

ABSTRACT Exchange proteins activated by cAMP (cyclic AMP) 2 (Epac2) is a guanine nucleotide exchange factor for Rap1, a small G protein involved in many cellular functions, including cell adhesion, differentiation, and exocytosis. Epac2 interacts with Ras-GTP via a Ras association (RA) domain. Previous studies have suggested that the RA domain was dispensable for Epac2 function. Here we show for the first time that Ras and cAMP regulate Epac2 function in a parallel fashion and the Ras-Epac2 interaction is required for the cAMP-dependent activation of endogenous Rap1 by Epac2. The mechanism for this requirement is not allosteric activation of Epac2 by Ras but the compartmentalization of Epac2 on the Ras-containing membranes. A computational modeling is consistent with this compartmentalization being a function of both the level of Ras activation and the affinity between Ras and Epac2. In PC12 cells, a well-established model for sympathetic neurons, the Epac2 signaling is coupled to activation of mitogen-activated protein kinases and contributes to neurite outgrowth. Taken together, the evidence shows that Epac2 is not only a cAMP sensor but also a bona fide Ras effector. Coincident detection of both cAMP and Ras signals is essential for Epac2 to activate Rap1 in a temporally and spatially controlled manner.

Regulation of the Small GTPase Rap1 and Extracellular Signal-Regulated Kinases by the Costimulatory Molecule CTLA-4

ABSTRACT The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) is activated following engagement of the T-cell receptor and is required for interleukin 2 (IL-2) production and T-cell proliferation. This activation is enhanced by stimulation of the coreceptor CD28 and inhibited by the coreceptor CTLA-4. We show that the small G protein Rap1 is regulated in the opposite manner; it is inhibited by CD28 and activated by CTLA-4. Together, CD3 and CTLA-4 activate Rap1 in a sustained manner. To delineate T-cell function in the absence of Rap1 activity, we generated transgenic mice expressing Rap1GAP1, a Rap1-specific GTPase-activating protein. Transgenic mice showed lymphadenopathy, and transgenic T cells displayed increased ERK activation, proliferation, and IL-2 production. More significantly, the inhibitory effect of CTLA-4 on T-cell function in Rap1GAP1-transgenic T cells was reduced. We demonstrate that CTLA-4 activates Rap1, and we propose that intracellular signals from CTLA-4 antagonize CD28, at least in part, at the level of Rap1.

The Interaction of Epac1 and Ran Promotes Rap1 Activation at the Nuclear Envelope

ABSTRACT Epac1 (exchange protein directly activated by cyclic AMP [cAMP]) couples intracellular cAMP to the activation of Rap1, a Ras family GTPase that regulates cell adhesion, proliferation, and differentiation. Using mass spectrometry, we identified the small G protein Ran and Ran binding protein 2 (RanBP2) as potential binding partners of Epac1. Ran is a small G protein best known for its role in nuclear transport and can be found at the nuclear pore through its interaction with RanBP2. Here we demonstrate that Ran-GTP and Epac1 interact with each other in vivo and in vitro. This binding requires a previously uncharacterized Ras association (RA) domain in Epac1. Surprisingly, the interaction of Epac1 with Ran is necessary for the efficient activation of Rap1 by Epac1. We propose that Ran and RanBP2 anchor Epac1 to the nuclear pore, permitting cAMP signals to activate Rap1 at the nuclear envelope.

Protein kinase a-dependent phosphorylation of Rap1 regulates its membrane localization and cell migration

Background: The small G protein Rap1 is phosphorylated within its carboxyl terminus by the cAMP-dependent protein kinase PKA. Results: This phosphorylation removes Rap1 from the plasma membrane to limit Rap1 signaling. Conclusion: Rap1 phosphorylation switches Rap1 off the membrane and terminates its activation. Significance: Carboxyl-terminal phosphorylation may be common among small G proteins to regulate GTP/GDP cycling and downstream signaling. The small G protein Rap1 can mediate “inside-out signaling” by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.

Phosphorylation of Rap1 by cAMP-dependent protein kinase (PKA) creates a binding site for KSR to sustain ERK activation by cAMP

Cyclic 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.

Protein Kinase A-independent Ras Protein Activation Cooperates with Rap1 Protein to Mediate Activation of the Extracellular Signal-regulated Kinases (ERK) by cAMP

Cyclic 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, with either Ras or Rap1 acting in distinct cell types. Using Hek293 cells, we show that both Ras and Rap1 are required for cAMP signaling to ERKs. The roles of Ras and Rap1 were distinguished by their mechanism of activation, dependence on the cAMP-dependent protein kinase (PKA), and the magnitude and kinetics of their effects on ERKs. Ras was required for the early portion of ERK activation by cAMP and was activated independently of PKA. Ras activation required the Ras/Rap guanine nucleotide exchange factor (GEF) PDZ-GEF1. Importantly, this action of PDZ-GEF1 was disrupted by mutation within its putative cyclic nucleotide-binding domain within PDZ-GEF1. Compared with Ras, Rap1 activation of ERKs was of longer duration. Rap1 activation was dependent on PKA and required Src family kinases and the Rap1 exchanger C3G. This is the first report of a mechanism for the cooperative actions of Ras and Rap1 in cAMP activation of ERKs. One physiological role for the sustained activation of ERKs is the transcription and stabilization of a range of transcription factors, including c-FOS. We show that the induction of c-FOS by cAMP required both the early and sustained phases of ERK activation, requiring Ras and Rap1, as well as for each of the Raf isoforms, B-Raf and C-Raf.