Maho Takahashi

Menin Missense Mutants Associated with Multiple Endocrine Neoplasia Type 1 Are Rapidly Degraded via the Ubiquitin-Proteasome Pathway

ABSTRACT MEN1 is a tumor suppressor gene that is responsible for multiple endocrine neoplasia type 1 (MEN1) and that encodes a 610-amino-acid protein, called menin. While the majority of germ line mutations identified in MEN1 patients are frameshift and nonsense mutations resulting in truncation of the menin protein, various missense mutations have been identified whose effects on menin activity are unclear. For this study, we analyzed a series of menin proteins with single amino acid alterations and found that all of the MEN1-causing missense mutations tested led to greatly diminished levels of the affected proteins in comparison with wild-type and benign polymorphic menin protein levels. We demonstrate here that the reduced levels of the mutant proteins are due to rapid degradation via the ubiquitin-proteasome pathway. Furthermore, the mutants, but not wild-type menin, interact both with the molecular chaperone Hsp70 and with the Hsp70-associated ubiquitin ligase CHIP, and the overexpression of CHIP promotes the ubiquitination of the menin mutants in vivo. These findings reveal that MEN1-causing missense mutations lead to a loss of function of menin due to enhanced proteolytic degradation, which may be a common mechanism for inactivating tumor suppressor gene products in familial cancer.

Coactivator-associated arginine methyltransferase 1, CARM1, affects pre-mRNA splicing in an isoform-specific manner.

Molecular diversity through alternative splicing is important for cellular function and development. However, little is known about the factors that regulate alternative splicing. Here we demonstrate that one isoform of coactivator-associated arginine methyltransferase 1 (named CARM1-v3) associates with the U1 small nuclear RNP-specific protein U1C and affects 5′ splice site selection of the pre-mRNA splicing. CARM1-v3 was generated by the retention of introns 15 and 16 of the primary transcript of CARM1. Its deduced protein lacks the C-terminal domain of the major isoform of CARM1 and instead has v3-specific sequences at the C terminus. CARM1-v3, but not the other isoforms, strongly stimulates a shift to the distal 5′ splice site of the pre-mRNA when the adenoviral E1A minigene is used as a reporter and enhances the exon skips in the CD44 reporter. A CARM1-v3 mutant lacking the v3-specific sequences completely lost the ability to regulate the alternative splicing patterns. In addition, CARM1-v3 shows tissue-specific expression patterns distinct from those of the other isoforms. These results suggest that the transcriptional coactivator can affect the splice site decision in an isoform-specific manner.

ERK5 activity is required for nerve growth factor-induced neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells

Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt2cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 μm, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt2cAMP, but BIX02188 and BIX02189, at 30 μm, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt2cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt2cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process.

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.

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.

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.

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.

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

Background: How the cAMP-dependent protein kinase PKA regulates B-Raf binding to Ras is not known. Results: PKA inhibits the binding of B-Raf to active Ras via phosphorylation of serine 365 in B-Raf. Conclusion: B-Raf can participate in the Ras-dependent activation of ERK in Ras-mutant cancers, and this is inhibited by PKA. Significance: PKA can block Ras binding to both Raf isoforms C-Raf and B-Raf. The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

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.

Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates

Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.