Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner

The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.The small GTPases of the Ras superfamily are involved in regulating many intracellular processes, including cell growth and division, cell morphology and movement, vesicular transport, and nuclear events (4, 40, 41). These proteins, which act as molecular switches to control various functions in the cell, are in the active, or “on,” state when bound to GTP and the inactive, or “off,” state when bound to GDP. The immediate control of these GTPase-mediated events resides in the proteins which regulate their GTP- or GDP-binding status. Two classes of regulatory proteins have been identified: the guanine nucleotide exchange factors (GEFs), whose physiological function is to convert GTPases from a GDP-bound state to a GTP-bound state, and the GTPase-activating proteins (GAPs), which turn off the GTPases by activating an intrinsic GTPase activity (3, 42, 44). The GEFs stimulate guanine nucleotide release to yield a GEF–apo-GTPase reaction intermediate and, in part because the GTP concentration in cells is higher than that of GDP, the formation of active GTP-bound GTPase is favored (61).Most of our understanding of the physical interaction of these regulatory molecules with the small GTPases is based on studies of the Ras protein (3, 42–44). For example, it is known that Ras GAPs bind to the effector loop of Ras (3, 42–44). The Ras effector loop, comprising residues 30 to 45, also interacts with the known downstream targets of Ras (42–44, 79).Numerous groups have contributed to the effort to identify Ras residues which are involved in interactions with GEFs. Residues 62 to 75 in the switch II region of H-ras were found to be involved, as were residues 103 and 105 in the alpha-helix 3–loop 7 (α3-L7) region (16, 38, 49, 57, 59, 60, 68, 69, 73). The effector loop (switch I region) of Ras was also implicated in direct interactions with GEFs (5, 38, 47, 79). The switch I, switch II, and α3-L7 regions of H-ras are found adjacent to each other on the surface of the molecule, as would be expected for a surface domain involved in GEF binding (see Fig. ​Fig.7)7) (36). The recently described crystal structure of H-ras complexed with Sos demonstrates that each of these three regions is indeed at the interface of the Ras-Sos complex (5).Open in a separate windowFIG. 7Diagram showing the structure of H-ras bound to GDP. The effector loop (residues 35 to 42) (magenta), switch II region (residues 62 to 76) (cyan blue), and α3-L7 region (residues 101 to 109) (green) of Ras are on the surface of the molecule and are located next to each other. The remaining H-ras structure is shown in yellow. GDP is shown in red. Two orientations of the molecules are presented: A and B show one orientation, and C and D show the other orientation. The locations of several amino acid residues of H-ras in regions involved in binding GEFs are indicated. The switch II region and the α3-L7 region are presented as either stick models (A and C) or space-filling models (B and D).Ras GEFs exhibit a modest preference for binding GDP-bound forms of Ras, whereas Ras GAPs preferentially bind GTP-bound forms (28, 37, 45, 49, 74). Thus, the GEFs and GAPs which affect the nucleotide-binding status of Ras preferentially bind their respective substrates rather than their products. The high affinities for substrates likely reflect structural differences between the two nucleotide-bound forms of Ras. Significantly, the switch I and switch II regions of H-ras, known to have altered structures when bound to either GDP or GTP, fall within the regions implicated in interactions with GEFs and GAPs (66).Recently, the crystal structure of the Sec7 domain of human Arno, a GEF for the Arf GTPase, and an analysis of the interaction sites of these two proteins have been reported (48). The analysis revealed that Arf interacts with its exchange factor in a manner reminiscent of the Ras interaction with its GEFs. Arf appears to use three noncontiguous segments of its polypeptide to interact with Sec7. Importantly, these three regions of the Arf protein are analogous to those used by Ras to interact with its GEFs. The switch I region (effector loop) and switch II region of Arf and Ras interact with their GEFs (5, 38, 47, 48, 79). Also, Ras residues 103 to 105 in the α3-L7 region and the corresponding residues of Arf (residues 113 to 115) appear to bind GEFs (5, 24, 48, 68, 69). While the GEF-binding sequences of Arf and Ras are at analogous positions in the GTPases, GEF-binding sequences of Ras do not show homology with the Arf sequences. The finding that these two distantly related GTPases use analogous regions to interact with their GEFs raises several questions relating to other subclasses of GTPases. For example, do the Rho and Rab/YPT1 families of GTPases interact with their GEFs by using domains analogous to those used by Ras and Arf? Do the different families of GEF use a similar mechanism for catalyzing guanine nucleotide exchange on small GTPases?We undertook the present study to ask whether other small GTPases use the regions corresponding to the GEF-binding domain of H-ras to interact with their cognate GEFs. For this study, we chose the yeast YPT1 protein, which is a member of the Rab family of small GTPases (22, 29, 70). This family of proteins is involved in regulating vesicular transport (54, 55). Previously we used a yeast genetic screen to identify Ras residues which were involved in binding to Ras GEFs (49). This screen uses both a dominant interfering mutant and a constitutively active mutant of Ras. Here we created analogous YPT1 mutants and demonstrated that they could be used in a similar genetic screen. We demonstrated that the mechanism of dominant interference of YPT1 mutant N22 (YPT1-N22) is sequestration of an endogenous essential GEF for YPT1 such that a lethal phenotype occurs because endogenous YPT1 cannot be activated. Using both site-directed and random mutagenesis procedures, we identified a series of intragenic suppressors of YPT1-N22, among which we predicted would be mutants which fail to sequester essential GEFs for YPT1 due to the loss of a complete GEF-binding domain.Among the intragenic suppressor mutations, we identified 10 residues, at positions 42, 43, 49, 69, 71, 73, 75, 107, 109, and 115, which were involved in in vitro binding to DSS4, a GEF which can stimulate nucleotide exchange on YPT1 in vitro (10, 50). The positions of these residues correspond to the switch I, switch II, and α3-L7 regions of Ras, the same regions found to be important for Ras interaction with GEFs.Our findings suggest that the interaction of Ras with its specific GEFs may prove to be a useful model for analyzing the structural basis underlying the interaction of other small GTPases with their cognate GEFs. Further, our findings, together with an analysis of the interactions of Ras and Arf GTPases with their GEFs, indicate that small GTPases of the Ras superfamily use similar regions for interactions with GEFs, suggesting a similar catalytic mechanism of guanine nucleotide exchange for all small GTPases.     

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.Internalization of epidermal growth factor receptors (EGFR)² and their subsequent delivery to lysosomes play key roles in attenuating EGF-mediated signaling cascades (1, 2). The proper delivery of EGFR into lysosomes for degradation requires a series of highly regulated targeting and delivery events. Following ligand binding, EGFR is internalized via endocytic vesicles that are subsequently targeted to early endosomes. This targeting event is mediated by the small GTPase, Rab5 (3, 4). Once delivered to the early endosome, receptors that are destined for degradation are incorporated into vesicles that bud into the lumen of the endosome, forming the multivesicular body (reviewed in Refs. 5, 6). Sequestration of the activated cytoplasmic domain of EGFR into the intralumenal vesicles of the multivesicular body effectively terminates receptor signaling (7). Subsequent fusion of the multivesicular body with lysosomes delivers the intralumenal vesicles and their contents into the lumen of the lysosome where they are degraded (reviewed in Refs. 8–10). Inactivating mutations in Rab5 disrupt the delivery of cell surface receptors, such as EGFR, to early endosomes, thereby inhibiting receptor trafficking to the lysosome and receptor degradation (11, 12). Therefore, activation of Rab5 is a key point of regulation for EGFR signaling.Rab5 cycles between an inactive GDP-bound state and an active GTP-bound state, and Rab5 activation requires the exchange of GDP to GTP. This exchange is catalyzed by guanine nucleotide exchange factors (GEFs) that are specific to the Rab5 family of proteins (reviewed in Ref. 13). Rab5 family GEFs all contain a catalytic vacuolar protein sorting 9 (Vps9) domain that facilitates the GDP to GTP exchange (14–17). Many Rab5 GEFs contain other functional domains that are involved in cell signaling events (13). Rin1 is a good example of a multidomain Rab5 GEF. In addition to the Vps9 domain, Rin1 also contains an Src homology 2 domain, a proline-rich domain, and a Ras association domain. Rin1 was originally identified through its ability to interact with active Ras (18), and a role for Rin1 in a number of cell signaling systems has been established, including EGF-mediated signaling (19–21). Rin1 directly interacts with the activated EGFR through its Src homology 2 domain (22). Furthermore, Ras occupation of the Rin1 Ras association domain positively impacts the Rab5 GEF activity of Rin1, which promotes EGFR internalization and attenuation in fibroblasts (23). However, Rin1 expression is up-regulated in several types of cancers, including squamous cell carcinoma (24), colorectal cancer (25), and cervical cancer (26), through duplications or rearrangements of the RIN1 locus. These studies suggest that Rin1 may also play a role in enhancing cell proliferation.It is well established that a large percentage of non-small cell lung adenocarcinomas exhibit up-regulation of EGFR and aberrant signaling through the Ras/MAPK pathway (reviewed in Ref. 27). In addition, a recent study examining 188 human lung adenocarcinomas identified that 132 of 188 tumor samples exhibited mutations relating to the Ras/MAPK signaling pathway (28). Accordingly, the role of Rin1 in non-small cell lung adenocarcinoma was addressed. Examination of a panel of non-small cell lung adenocarcinoma lines (including A549) revealed enhanced Rin1 expression relative to a nontransformed lung epithelial cell line (BEAS-2B). Depletion of Rin1 from A549 cells resulted in decreased proliferation. This decrease correlated with a reduction in EGF-activated ERK phosphorylation and the stabilization of cell surface EGFR. These defects were complemented by wild type Rin1 expression but not by mutant Rin1 lacking a functional Vps9 domain, suggesting that the GEF activity of Rin1 is necessary for proper EGFR signaling in A549 cells. In addition, overexpression of Rin1Δ, dominant negative Rab5, and dynamin resulted in similar defects in cell proliferation and EGFR signaling as Rin1 depletion. These data indicate that proper EGFR internalization and trafficking are critical for robust EGFR-mediated signaling and cell proliferation in A549 cells and offer evidence that Rin1 positively regulates cell proliferation in non-small cell lung adenocarcinoma.     

Requirement of Myosin Vb��Rab11a��Rab11-FIP2 Complex in Cholesterol-regulated Translocation of NPC1L1 to the Cell Surface

Niemann-Pick C1-like 1 (NPC1L1) plays a critical role in the enterohepatic absorption of free cholesterol. Cellular cholesterol depletion induces the transport of NPC1L1 from the endocytic recycling compartment to the plasma membrane (PM), and cholesterol replenishment causes the internalization of NPC1L1 together with cholesterol via clathrin-mediated endocytosis. Although NPC1L1 has been characterized, the other proteins involved in cholesterol absorption and the endocytic recycling of NPC1L1 are largely unknown. Most of the vesicular trafficking events are dependent on the cytoskeleton and motor proteins. Here, we investigated the roles of the microfilament and microfilament-associated triple complex composed of myosin Vb, Rab11a, and Rab11-FIP2 in the transport of NPC1L1 from the endocytic recycling compartment to the PM. Interfering with the dynamics of the microfilament by pharmacological treatment delayed the transport of NPC1L1 to the cell surface. Meanwhile, inactivation of any component of the myosin Vb·Rab11a·Rab11-FIP2 triple complex inhibited the export of NPC1L1. Expression of the dominant-negative mutants of myosin Vb, Rab11a, or Rab11-FIP2 decreased the cellular cholesterol uptake by blocking the transport of NPC1L1 to the PM. These results suggest that the efficient transport of NPC1L1 to the PM is dependent on the microfilament-associated myosin Vb·Rab11a·Rab11-FIP2 triple complex.Cholesterol homeostasis in human bodies is maintained through regulated cholesterol synthesis, absorption, and excretion. Intestinal cholesterol absorption is one of the major pathways to maintain cholesterol balance. NPC1L1 (Niemann-Pick C1-like protein 1), a polytopic transmembrane protein highly expressed in the intestine and liver, is required for dietary cholesterol uptake and biliary cholesterol reabsorption (1–4). Genetic or pharmaceutical inactivation of NPC1L1 significantly inhibits cholesterol absorption and confers the resistance to diet-induced hypercholesterolemia (1, 2, 4). Ezetimibe, an NPC1L1-specific inhibitor, is currently used to prevent and treat cardiovascular diseases (5).Human NPC1L1 contains 1,332 residues with 13 transmembrane domains (6). The third to seventh transmembrane helices constitute a conserved sterol-sensing domain (4, 7). NPC1L1 recycles between the endocytic recycling compartment (ERC)³ and the plasma membrane (PM) in response to the changes of cholesterol level (8). ERC is a part of early endosomes that is involved in the recycling of many transmembrane proteins. It is also reported that ERC is a pool for free cholesterol storage (9). When cellular cholesterol concentration is low, NPC1L1 moves from the ERC to the PM (8, 10). Under cholesterol-replenishing conditions, NPC1L1 and cholesterol are internalized together and transported to the ERC (8). Disruption of microfilament, depletion of the clathrin·AP2 complex, or ezetimibe treatment can impede the endocytosis of NPC1L1, thereby decreasing cholesterol internalization (8, 10, 11).The microfilament (MF) system, part of the cytoskeleton network, is required for multiple cellular functions such as cell shape maintenance, cell motility, mitosis, protein secretion, and endocytosis (12, 13). The major players in the microfilament system are actin fibers and motor proteins (14). Actin fibers form a network that serves as the tracks for vesicular transport (15, 16). Meanwhile, the dynamic assembly and disassembly of actin fibers and the motor proteins provides the driving force for a multitude of membrane dynamics including endocytosis, exocytosis, and vesicular trafficking between compartments (15, 16).Myosins are a large family of motor proteins that are responsible for actin-based mobility (14). Class V myosins (17, 18), comprising myosin Va, Vb, and Vc, are involved in a wide range of vesicular trafficking events in different mammalian tissues. Myosin Va is expressed mainly in neuronal tissues (19, 20), whereas myosins Vb and Vc are universally expressed with enrichment in epithelial cells (21, 22). Class V myosins are recruited to their targeting vesicles by small GTPase proteins (Rab) (23). Rab11a and Rab11 family-interacting protein 2 (Rab11-FIP2) facilitate the binding of myosin Vb to the cargo proteins of endocytic recycling vesicles (24–28).Myosin Vb binds Rab11a and Rab11-FIP2 through the C-terminal tail (CT) domain. The triple complex of myosin Vb, Rab11a, and Rab11-FIP2 is critical for endocytic vesicular transport and the recycling of many proteins including transferrin receptor (29), AMPA receptors (30), CFTR (28), GLUT4 (31, 32), aquaporin-2 (26), and β2-adrenergic receptors (33). The myosin Vb-CT domain (24) competes for binding to Rab11a and Rab11-FIP2 and functions as a dominant-negative form. Expression of the CT domain substantially impairs the transport of vesicles. Deficient endocytic trafficking is also observed in cells expressing the GDP-locked form of Rab11a (S25N) (34) or a truncated Rab11-FIP2, which competes for the rab11a binding (35).Here we investigated the roles of actin fibers and motor proteins in the cholesterol-regulated endocytic recycling of NPC1L1. Using pharmaceutical inactivation, dominant-negative forms, and an siRNA technique, we demonstrated that actin fibers and myosin Vb·Rab11a·Rab11-FIP2 triple complex are involved in the export of NPC1L1 to the PM and that this intact MF-associated triple complex is required for efficient cholesterol uptake. Characterization of the molecules involved in the recycling of NPC1L1 may shed new light upon the mechanism of cholesterol absorption.     

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcγ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcγ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (FcγR)⁴ mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of FcγR on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin- and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, cross-linking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, FcγR possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho- and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase Cγ (PLCγ), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for FcγR-induced phagocytosis (7, 8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) undergoes a transient accumulation at the phagocytic cup, which is rapidly superseded by its complete elimination from the nascent phagosome (7). The secondary disappearance of PtdIns(4,5)P₂ is attributable in part to the localized generation of phosphatidylinositol 3,4,5-trisphosphate, which has been reported to accumulate at sites of phagocytosis (9). Activation of PLCγ is also believed to contribute to the acute disappearance of PtdIns(4,5)P₂ in nascent phagosomes. Indeed, the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate has been detected by chemical means during FcγR-evoked particle ingestion (10, 11). Moreover, imaging experiments revealed that DAG appears at the time and at the precise site where PtdIns(4,5)P₂ is consumed (7).Two lines of evidence suggest that the DAG generated upon engagement of phagocytic receptors modulates particle engulfment. First, antagonists of PLC severely impair phagocytosis by macrophages (7, 12). This inhibition is not mimicked by preventing the associated [Ca²⁺] transient, suggesting that DAG, and not inositol 1,4,5-trisphosphate, is the crucial product of the PLC (13). Second, the addition of exogenous DAG or phorbol esters, which mimic the actions of endogenous DAG, augment phagocytosis (14, 15).Selective recognition of DAG by cellular ligands is generally mediated by specific regions of its target proteins, called C1 domains (16). Proteins bearing C1 domains include, most notably, members of the classical and novel families of protein kinase C (PKC), making them suitable candidates to account for the DAG dependence of phagocytosis. Indeed, PKCα, a classical isoform, and PKCϵ and PKCδ, both novel isoforms, are recruited to phagosomes (12, 15, 17, 18). Although the role of the various PKC isoforms in particle engulfment has been equivocal over the years, Cheeseman et al. (12) convincingly demonstrated that PKCϵ contributes to particle uptake in a PLC- and DAG-dependent manner.PKCs are not the sole proteins bearing DAG-binding C1 domains. Similar domains are also found in several other proteins, including members of the RasGRP family, chimaerins, and Munc-13 (19–21). One or more of these could contribute to the complex set of responses elicited by FcγR-induced DAG production. The RasGRP proteins are a class of exchange factors for the Ras/Rap family of GTPases (22). There are four RasGRP proteins (RasGRP1 to -4), and emerging evidence has implicated RasGRP1 and RasGRP3 in T and B cell receptor signaling (23–27).The possible role of DAG-mediated signaling pathways other than PKC in phagocytosis and the subsequent inflammatory response has not been explored. Here, we provide evidence that DAG stimulates Ras and Rap1 at sites of phagocytosis, probably through RasGRPs. Last, the functional consequences of Ras and Rap1 activation were analyzed.     

Down-regulation of Ras-related Protein Rab 5C-dependent Endocytosis and Glycolysis in Cisplatin-resistant Ovarian Cancer Cell Lines

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer.Ovarian cancer is the major cause of death in women with gynecological cancer. Early diagnosis of ovarian cancer is difficult, while its progression is fast. The standard treatment is surgical removal followed by platinum-taxane chemotherapy. However, the efficacy of the traditional surgery and chemotherapy is rather compromised and platinum resistant cancer recurs in ∼25% of patients within six months, and the overall five-year survival rate is about 31% (1–3). Virtually no efficient second line treatment is available. In order to increase survival rates from ovarian cancer and enhance patients'' quality of life, new therapeutic targets are urgently required, necessitating a deeper understanding of molecular mechanisms of drug resistance.Mechanisms of drug-resistance in ovarian cancer have been extensively studied over the last 30 years. Earlier studies have found that multiple factors are linked to drug resistance in human ovarian cancer including reduced intracellular drug accumulation, intracellular cisplatin inactivation, and increased DNA repair (4). Reduced cellular drug accumulation is mediated by the copper transporter-1 responsible for the influx of cisplatin (5–9) and MDR1, which encodes an integral membrane protein named P-glycoprotein for the active efflux of platinum drugs. Up-regulation of MDR1 has been observed in cisplatin-treated ovarian cancer cells although cisplatin is not a substrate of P-glycoprotein (10–13). A fraction of intracellular cisplatin can be converted into cisplatin-thiol conjugates by glutathione-S-transferase (GST) π, leading to inactivation of cisplatin. Up-regulation of both GSTπ and γ-glutamylcysteine synthetase has been associated with cisplatin resistance in ovarian, cervical and lung cancer cell lines (14–18). Binding of cisplatin to DNA leads to intrastrand or interstrand cross-links that alter the structure of the DNA molecule causing DNA damage. It has been amply documented that pathways for recognition and repair of damaged DNA are up-regulated in drug-resistant cancer cells (19–26). Furthermore, the secondary mutations have been identified, which restore the wild-type BRCA2 reading frame enhancing the acquired resistance to platinum-based chemotherapy (24). Alternations in other signaling pathways have also been found in drug resistant ovarian cancer (27–29). For example, DNA-PK phosphorylates RAC-alpha serine/threonine-protein kinase (AKT) and inhibits cisplatin-mediated apoptosis (28); and silencing of HDAC4 increases acetyl-STAT1 levels to prevent platinum-induced STAT1 activation and restore cisplatin sensitivity (29).Proteomics is playing an increasingly important role in identifying differentially expressed proteins between drug-resistant and drug sensitive ovarian cancer cells (30–35). An earlier study has identified 57 differentially expressed proteins in human ovarian cancer cells and their platinum-resistant sublines, including annexin A3, destrin, cofilin 1, Glutathione-S-transferase omega 1, and cytosolic NADP+-dependent isocitrate dehydrogenase using 2D gel electrophoresis (30). Employing a similar 2D gel electrophoresis approach, changes in protein expressions of capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3, and phosphoglycerate kinase 1 (PGK)¹ were found in A2780 and drug-resistant A2780 cells (32). It is worth mentioning that ALDO and PGK are glycolytic enzymes, indicating that glycolysis plays a role in drug resistance. Studies have demonstrated that resistance to platinum drugs in ovarian cancer cells is linked to mitochondrial dysfunctions in oxidative phosphorylation and energy production (36–40). Mitochondrial proteomic analysis of drug-resistant cells has shown that five mitochondrial proteins (ATP-a, PRDX3, PHB, ETF, and ALDH) that participate in the electron transport respiratory chain are down-regulated in drug-resistant cell lines (41). PRDX3 is involved in redox regulation of the cell to protect radical-sensitive enzymes from oxidative damage. However, it is not clear how down-regulation of PRDX3 is associated with drug-resistance. A more recent study showed that activated leukocyte cell adhesion molecule (ALCA) and A kinase anchoring protein 12 (AKAP12) are elevated in drug-resistant A2780-CP20 cells by quantifying the mitochondrial proteins (42). Despite these efforts, the drug-resistance mechanisms are not yet well understood.In this work, we established and characterized a drug-resistant cell line A2780-DR from A2780 cells. We employed a quantitative proteomic method to identify the differentially expressed proteins between A2780 and A2780-DR cells. Expression changes of selected proteins were confirmed by qPCR and Western blotting. We also used shRNA silencing to explore functions of Rab 5C and Rab 11B proteins in drug resistance. Our data indicate that the differentially expressed proteins participate in a variety of cellular processes and enhance our understanding of the mechanisms of drug resistance in ovarian cancer cells.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

The Vac14-interaction Network Is Linked to Regulators of the Endolysosomal and Autophagic Pathway

The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositol-modifying enzymes are tightly interconnected.Lipid kinases and phosphatases tightly regulate the interconversion and abundance of different phosphoinositide lipid derivatives (PIPs),¹ which are crucial components for the identity of eukaryotic membranes (1, 2). PIPs and their modifying proteins control multiple cellular functions such as signal transduction, cytoskeletal dynamics, and membrane trafficking (1, 2). Synthesis and turnover of the low-abundant lipid phosphatidylinositol-3,5-bisphosphate is controlled by the Vac14 complex. This complex contains the scaffold protein Vac14 (ArPIKfyve), the lipid kinase PIKfyve (Fab1), and its counteracting lipid phosphatase FIG4 (SAC3) (3, 4).The overall protein structure of Vac14 is conserved from yeast to human and consists of multiple tandem HEAT (huntingtin, elongation Factor 3, PR65/A, TOR) repeats and a rod-like helical domain that controls protein–protein interactions (5, 6). Previous studies showed that the aminoterminal part of Vac14 mediates the binding to PIKfyve. The carboxyterminal region is crucial for the interaction with FIG4 and also contains a PDZ-binding motif, which binds to nNOS (5, 7–11). Moreover, Vac14-oligomerization, which is essential for the nucleation of an active Vac14-complex, overlaps with the FIG4 interaction binding site (5, 6, 12, 13).The lack of Vac14 or the ectopic overexpression of a PIKfyve binding-deficient Vac14 mutant in vivo results in a reduced level of phosphatidylinositol-3,5-bisphosphate and is accompanied by enhanced formation of enlarged intracellular vesicles, hereinafter called vacuoles (5, 14, 15). This was initially observed in yeast, where it leads to enlargement of the yeast vacuole, which is comparable to the mammalian lysosome (12, 16, 17). The vacuoles in mammalian cells are heterogeneous, positive for early or late endosomal structures, and involved in vesicular trafficking processes from the late endosome to the trans-Golgi network (14, 18, 19).We now report that in addition to overexpression of the PIKfyve binding deficient mutant, overexpression of the Vac14 wild type was sufficient to induce vacuolization in human cell cultures. The Vac14-based vacuolization leads to a significant accumulation of predominantly late endosomal and autophagosomal marker proteins with only late endosomal proteins decorating the vacuolar membranes. These results almost completely phenocopy previously described effects of the inhibitor YM201636, which specifically blocks PIKfyve lipid kinase activity (20–24).To identify proteins involved in Vac14-induced vacuolization, we used a protein affinity approach combined with multidimensional protein identification technology (MudPIT). The evaluation of the identified peptides elucidated numerous potential Vac14 interacting proteins involved in intracellular trafficking and membrane dynamics, with Rab9 and TBC1D15 being the most promising candidates. The specificity of the Vac14 interaction with Rab9 and the Rab7 regulator TBC1D15 was confirmed by co-immunoprecipitation assays. Moreover, we were able to demonstrate that Rab9 accumulates during vacuolization and localizes on the limiting membranes of vacuoles as a result of Vac14 overexpression. In summary, the identification of Rab9 and TBC1D15 as novel interaction partners provides new insights into the molecular functions of Vac14 in vesicular transport processes.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Diacylglycerol Kinase �� Augments C-Raf Activity and B-Raf/C-Raf Heterodimerization

The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase η (DGKη) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKη1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKη serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKη-silenced HeLa cells, this study revealed that DGKη activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKη enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKη physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKη-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKη acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.The Ras/Raf/MEK³/ERK signaling pathway is critical for the transduction of the extracellular signals to the nucleus, regulating diverse physiological processes such as cell proliferation, differentiation, and survival (1, 2). The binding of extracellular ligands, such as growth factors and cytokines, to cell surface receptors activates Ras. The Raf serine/threonine kinase transmits signals from activated Ras to the downstream protein kinases, MEK1 and MEK2, subsequently leading to activation of ERK1 and ERK2.In mammals, the Raf kinase consists of three isoforms, A-Raf, B-Raf, and C-Raf (Raf-1). It is clinically known that both B-Raf and C-Raf mutations are associated with human cancers (3–5). Knock-out mouse studies demonstrated that each individual Raf isoform has distinct functions, although the three Raf isoforms have high homology in the amino acid sequence (6). The mechanisms underlying C-Raf activation are complicated and thus are not completely understood (3). In response to extracellular signals, C-Raf is initially recruited from cytosol to the plasma membrane and undergo conformational changes by binding directly to the active Ras (7). In addition, other modifications and factors are required for the sufficient activation of C-Raf. For example, dephosphorylation of Ser-259 and phosphorylation of Ser-338, Tyr-341, Thr-491, and Ser-494 are critical for the activation of C-Raf (8–11). Feedback phosphorylation of C-Raf by ERK was also reported to be important for the modulation of C-Raf activity (12, 13). C-Raf activity is regulated by the interaction with 14-3-3 protein (14). Moreover, the heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf, has been reported to play an essential role in the activation of the MEK-ERK signaling pathway (15–17). Although B-Raf and C-Raf are the central regulatory components in the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade involved in a variety of pathophysiological events, the activation mechanisms of C-Raf by B-Raf are still unclear.Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol to generate phosphatidic acid. DGK has been recently recognized as an emerging key regulator in a wide range of cell signaling systems (18–20). To date, 10 mammalian DGK isozymes have been identified. They characteristically contain two or three protein kinase C-like C1 domains and a catalytic region and are subdivided into five subtypes according to their structural features (18–20). Their structural variety and distinct expression patterns in tissues allow us to presume that each DGK isozyme has its own biological functions. Indeed, recent studies have revealed that individual DGK isozymes play distinct roles in cell functions through interactions with unique partner proteins such as protein kinase C (21, 22), Ras guanyl nucleotide-releasing protein (23, 24), phosphatidylinositol-4-phosphate 5-kinase (25), chimerins (26, 27), AP-2 (28), and PSD-95 (29).DGKη belongs to the type II DGKs containing a pleckstrin homology domain at the N terminus and the separated catalytic region (19, 30). Two alternative splicing products of DGKη have been identified as DGKη1 and -η2 (31). DGKη2 possesses a sterile α-motif (SAM) domain at the C terminus, whereas DGKη1 does not. This study demonstrated that the expression levels of DGKη1 and -η2 were regulated differently by glucocorticoid, and that they were translocated from the cytoplasm to endosomes in response to stress stimuli as osmotic shock and oxidative stress (31). However, the physiological roles of DGKη remain unknown.This study showed that siRNA-dependent knockdown of DGKη inhibits cell proliferation of the HeLa cells. In addition, DGKη is required for the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade activated by epidermal growth factor (EGF). Intriguingly, DGKη regulates recruitment of B-Raf and C-Raf from cytosol to membranes and their heterodimerization. Moreover, this study demonstrated that DGKη activates C-Raf but not B-Raf in an EGF-dependent manner. The data show DGKη as a novel key regulator of the Ras/B-Raf/C-Raf/MEK/ERK signaling pathway.     

Nucleophosmin and Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal Transduction

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.Ras proteins are small GTPases that function as molecular switches on the inner leaflet of the plasma membrane, conveying extracellular signals to the cell interior. Ras proteins are critical regulators of signal transduction pathways controlling key cell fates such as cell growth, differentiation, and apoptosis. Deregulation of these pathways results in aberrant cell growth and tumor formation. Mutations that render Ras constitutively active are found in ∼15% of human cancers, making Ras one of the most clinically significant proteins in human carcinogenesis. Oncogenic mutations are most prevalent in the K-Ras gene, accounting for a large proportion of solid tumors including 90% of pancreatic cancer, 50% of colon cancer, and 30% of non-small cell lung cancer (1, 2).The three major Ras isoforms, H-, N-, and K-Ras generate distinct signal outputs in intact cells, signifying specific roles for each isoform. These functional differences stem from significant sequence divergence in the Ras C-terminal 25 amino acids of the hypervariable region (HVR)³ that directs post-translation attachment of different lipid anchors. The minimal membrane anchor of H-Ras comprises two palmitate groups and a farnesyl group, whereas K-Ras is tethered by a farnesyl group and a polybasic domain (3, 4). These minimal anchors, together with flanking protein sequences and the G-domain, interact with lipids and proteins of the plasma membrane, driving the Ras isoforms into spatially and structurally distinct nanodomains on the plasma membrane (5, 6). Ras lateral segregation is further modulated by the activation state of Ras; active GTP-loaded H-Ras is organized in cholesterol-independent nanoclusters, whereas inactive GDP-loaded H-Ras is arrayed in cholesterol-dependent nanoclusters (5, 7–9). Recent work has also shown that GTP-K-Ras clusters into nanodomains that are spatially distinct from GDP-K-Ras, although both types of nanocluster are cholesterol-independent and actin-dependent (7, 9). K-Ras-GTP nanoclustering, however, is regulated by galectin-3, which operates as a nanodomain scaffold (10, 11).Ras-GTP nanoclusters are the sites of Raf/MEK and ERK recruitment to the plasma membrane. Scaffolding all components of the MAPK module within nanoclusters rewires the biochemistry to generate a digital ERKpp output. The operation of Ras-GTP nanoclusters as highly sensitive digital switches is critical to deliver high fidelity signal transmission across the plasma membrane (12–14). A key parameter in epidermal growth factor (EGF) receptor to MAPK signal transmission is the fraction of Ras-GTP that forms nanoclusters; this clustered fraction sets the gain for cellular MAPK signaling (15, 16).NPM (also known as B23) and nucleolin are multifunctional phosphoproteins predominately localized to the nucleolus that play key roles in ribosome biogenesis (17–19). For example, NPM exhibits ribonuclease activity and preferentially cleaves pre-rRNA. NPM and nucleolin also have functions outside of the nucleolus. Both proteins shuttle between the nucleolus and the cytoplasm (20), and this shuttling may allow NPM to operate as molecular chaperone (21). In addition cytosolic NPM is involved in centrosome duplication (22). Like Ras proteins, NPM and nucleolin regulate cell proliferation and transformation and are overexpressed in multiple cancers (23). However, the physiological role of NPM in carcinogenesis remains controversial because it has been described as both an oncogene and a tumor suppressor (23).In this study we identify NPM and nucleolin as proteins that interact specifically with K-Ras but not H-Ras. Furthermore we definitively identify a subset of NPM and nucleolin on the inner leaflet of the plasma membrane where both proteins interact with K-Ras. Importantly, NPM and nucleolin stabilize K-Ras levels on the plasma membrane, leading to an increase in the K-Ras clustered fraction, which amplifies signal output from the MAPK pathway. Combined, our data indicate that NPM and nucleolin play a critical role in signal transduction via the MAPK pathway.     

Systems-wide Analysis of K-Ras,Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.The Ras oncoproteins are small monomeric GTPases that transduce mitogenic signals from cell surface receptor tyrosine kinases (RTKs) to intracellular serine/threonine kinases. Approximately thirty percent of human tumors harbor a somatic gain-of-function mutation in one of three RAS genes, resulting in the constitutive activation of Ras signaling and the aberrant hyperactivation of growth-promoting effector pathways (1). Designing therapeutic agents that directly target Ras has been challenging (2, 3), and thus clinical development efforts have focused on targeting effector pathways downstream of Ras. The Raf-MEK-ERK and PI3K-Akt effector pathways have been extensively studied and several small molecule inhibitors targeting these pathways are currently under clinical evaluation (4, 5). However, biochemical studies and mouse models indicate that several additional effector pathways are essential for Ras-driven transformation and tumorigenesis (6–11). Hence, a comprehensive characterization of these effector pathways may reveal additional druggable targets.The Rho GTPase Cdc42 lies downstream of Ras (12–14) and regulates many cellular processes that are commonly perturbed in cancer, including migration, polarization, and proliferation (15) (Fig. 1A). Importantly, Cdc42 is overexpressed in several types of human cancer (16–20) and is required for Ras-driven cellular transformation (13, 21, 22). Recent studies show that genetic ablation of Cdc42 impairs Ras-driven tumorigenesis (13), indicating the potential of Cdc42 and its effectors as drug targets in Ras mutant tumors.Open in a separate windowFig. 1.Experimental workflow. A, K-Ras is a small GTPase that regulates the activity of a variety of downstream proteins including the Rho GTPase Cdc42. The PAK4 serine/threonine kinase is a direct effector of Cdc42 and regulates actin reorganization, microtubule stability, and cell polarity. B, To measure large-scale phosphorylation changes induced by constitutive K-Ras or Cdc42 signaling or PAK4 ablation, the quantitative label-free PTMscan® approach was employed (Cell Signaling Technology). Briefly, for each condition extracted proteins were digested with trypsin and separated from non-peptide material by solid-phase extraction with Sep-Pak C18 cartridges. Three phosphorylation motif antibodies were used serially to isolate phosphorylated peptides in independent immunoaffinity purifications (CDK substrate motif [K R]-pS-P-X-[K R], CK substrate motif pT-[D E]-X-[D E], PKD substrate motif l-X-R-X-X-p[S T]). The samples were run in duplicate and tandem mass spectra were collected with an LTQ-Orbitrap hybrid mass spectrometer. pLPC is an empty vector control.In particular, the p21-activated kinases (PAKs) are Cdc42 effectors that have generated significant interest (23, 24), as they are central components of key oncogenic signaling pathways and regulate cytoskeletal organization, cell migration, and nuclear signaling (25). The PAK family is comprised of six members and is subdivided into two groups (Groups I and II) based on sequence and structural homology. Group I PAKs (PAK1–3) are relatively well characterized, however, much less is known regarding the function and regulation of Group II PAKs (PAK4–6). The kinase domains of Group I and II PAKs share only about 50% identity, suggesting the two groups may recognize distinct substrates and govern unique cellular processes (26).The Group II PAK family member PAK4 is of particular interest as it is overexpressed or genetically amplified in several lung, colon, prostate, pancreas, and breast tumor cell lines and samples (26–30). Furthermore, functional studies have implicated PAK4 in cell transformation, cell invasion, and migration (27, 31). Xenograft studies in athymic mice show an important role for PAK4 in mediating Cdc42- or K-Ras-driven tumor formation, highlighting a critical role for Pak4 downstream of these GTPases (32). Given its roles in transformation, tumorigenesis, and oncogenic signaling, there is significant interest in targeting PAK4 therapeutically (23). PAK4 binds and phosphorylates several proteins involved in cytoskeletal organization and apoptosis, including Lim domain kinase 1 (LIMK1) (33), guanine nucleotide exchange factor-H1 (GEF-H1) (34), Raf-1 (35), and Bad (36). However, the Group I PAK family member PAK1 also phosphorylates several of these PAK4 targets (37). Thus, there remains a need to identify robust and selective pharmacodynamic biomarkers for PAK4 inhibition.Despite the importance of PAK4 and its upstream regulators in cancer development, few studies have sought to comprehensively characterize the spectrum of K-Ras, Cdc42, or PAK4 mediated phosphorylation signaling (37–39). Recent developments in mass spectrometry allow the in-depth identification and quantitation of thousands of phosphorylation sites (40–43). The majority of large-scale efforts have aimed to identify the basal phosphoproteomes of different species (44, 45) or tissues (46) to characterize global steady-state phosphorylation. However, this methodology can also be applied to quantify perturbed phosphorylation regulation in cancer signaling pathways (40, 47–49), and has the potential to reveal novel biomarkers of oncogenic signaling.In this study, we completed a label-free quantitative analysis of K-Ras, Cdc42, and PAK4 phosphorylation signaling using the PTMScan® method, which has proven as robust and reproducible quantitation technology (50, 51). We quantified phosphorylation levels in wild-type and PAK4 knockout NIH3T3 cells expressing oncogenic K-Ras, activated Cdc42, or an empty vector control to elucidate the molecular pathways and functions modulated by these key signaling proteins. We report relative quantitation of 2152 phosphorylated peptides on 1062 proteins among the different conditions, and find that many of the regulated phosphoproteins are associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. To our knowledge, our study is the first to examine the overlap among signaling networks regulated by K-Ras, Cdc42, and PAK4, and provides a resource for future studies to further interrogate the perturbation of this signaling pathway.     

Mapping of Vps21 and HOPS Binding Sites in Vps8 and Effect of Binding Site Mutants on Endocytic Trafficking

Vps8 is a subunit of the CORVET tethering complex, which is involved in early-to-late endosome fusion. Here, we examine the role of Vps8 in membrane fusion at late endosomes in Saccharomyces cerevisiae. We demonstrate that Vps8 associates with membranes and that this association is independent of the class C/HOPS core complex and, contrary to a previous report, also independent of the Rab GTPase Vps21. Our data indicate that Vps8 makes multiple contacts with membranes. One of these membrane binding regions could be mapped to the N-terminal part of the protein. By two-hybrid analysis, we obtained evidence for a physical interaction between Vps8 and the Rab5 homologue Vps21. In addition, the interaction with the HOPS core complex was confirmed by immunoprecipitation experiments. By deletion analysis, the Vps21 and HOPS binding sites were mapped in Vps8. Deletions that abrogated HOPS core complex binding had a strong effect on the turnover of the endocytic cargo protein Ste6 and on vacuolar sorting of carboxypeptidase Y. In contrast, deletions that abolished Vps21 binding showed only a modest effect. This suggests that the Vps21 interaction is not essential for endosomal trafficking but may be important for some other aspect of Vps8 function.The compartments of the exocytic/endocytic membrane system are dynamic structures that continuously exchange materials by budding and fusion of transport vesicles. Despite this continuous exchange, the compartments maintain their specific identities. A basic machinery consisting of tethering factors, Rab GTPases, SNARE proteins, and Sec1/Munc18 (SM) proteins accomplishes membrane targeting and fusion. For each individual membrane fusion event, a characteristic set of proteins is used.We are interested in a particular membrane fusion step, the fusion of early endosome-derived vesicles with late endosomes. Screening for vps (vacuolar protein sorting) mutants in Saccharomyces cerevisiae identified factors involved in this fusion step (3). Mutants defective in the early-to-late endosome trafficking step belong to the class D group of vps mutants, whose hallmark is an enlarged vacuole (21). Among the class D functions, representatives of the main groups of targeting and fusion factors can be found. The Q-SNARE protein Pep12, for instance, a member of the syntaxin family, serves as a marker for late endosomal membranes (2). Together with the Q-SNAREs Vti1 and Syn8 or Tlg1, it forms two alternative t-SNARE complexes on late endosomal membranes (17). These t-SNAREs combine with the v-SNARES Snc1/Snc2 or Ykt6 to form functional trans-SNARE complexes. Pep12 functionally interacts with another class D protein, the SM protein Vps45 (4). Another component of the basic fusion machinery at late endosomes is the class D protein Vps21, a member of the Rab GTPase family and the yeast homologue of mammalian Rab5 (8, 12, 30). Rab proteins are key regulators of membrane fusion (9). They are involved in the recruitment of tethering and docking factors, and by their interplay with Rab effectors they contribute to the establishment of specific membrane domains. Another class D protein connected to Rab function is Vps9, a guanidine nucleotide exchange factor (GEF) for Vps21 (11).Additional class D proteins are involved in vesicle tethering at late endosomes. Basically, there are two kinds of tethers, proteins containing extensive coiled-coil domains and large multisubunit complexes (33). The prototype of the coiled-coil tethers is p115, with its yeast homologue Uso1, involved in tethering of vesicles to Golgi apparatus membranes (25). Another member of this class is EEA1, which is involved in tethering of vesicles to endosomes. The yeast class D protein Vps19/Pep7/Vac1 could be functionally similar to EEA1 (16). Two further class D proteins, Vps3 and Vps8, are part of the multisubunit (class C core vacuole/endosome tethering) CORVET tethering complex (20, 32). This complex shares core components with the HOPS (homotypic fusion and vacuole protein sorting) tethering complex involved in homotypic vacuolar fusion (28). This core complex, the class C Vps complex, consists of Vps11/Pep5, Vps16, Vps18/Pep3, and the SM protein Vps33 (26). Instead of Vps3 and Vps8, HOPS contains two additional subunits, Vps39/Vam6 and Vps41 (35), which appear to be functionally equivalent to Vps3 and Vps8 (20). In addition to bridging donor and acceptor membranes, tethers appear to be involved in coordinating Rab and SNARE functions. This was suggested by the finding that the equivalent CORVET/HOPS subunits Vps3 and Vps39/Vam6 both display GEF activity toward their respective Rab proteins, Vps21 and Ypt7 (20, 35). In addition, whole tethering complexes act as Rab effectors by binding to activated Rab-GTP and interact with the corresponding SNARE complexes (6, 20, 31).How exactly the tethers coordinate Rab and SNARE functions during membrane fusion is at present unclear. Here, we examine the function of the CORVET subunit Vps8 (5, 13) in membrane fusion at late endosomes in yeast. We demonstrate that Vps8 directly associates with membranes. Contrary to a previous report (13), we show that this membrane association is not dependent on Vps21. We further investigate the functional relationship between Vps8 and Vps21. We found that Vps21 physically interacts with Vps8 but that this interaction does not appear to be absolutely required for endosomal trafficking. Finally, we speculate that Vps8 could be part of a higher-order structure.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis

Exocytosis of the acrosome (the acrosome reaction) relies on cAMP production, assembly of a proteinaceous fusion machinery, calcium influx from the extracellular medium, and mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in a protein kinase A- and extracellular calcium-independent manner. The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap. We show here that a soluble adenylyl cyclase synthesizes the cAMP required for the acrosome reaction. Epac stimulates the exchange of GDP for GTP on Rap1, upstream of a phospholipase C. The Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP induces a phospholipase C-dependent calcium mobilization in human sperm suspensions. In addition, our studies identify a novel connection between cAMP and Rab3A, a secretory granule-associated protein, revealing that the latter functions downstream of soluble adenylyl cyclase/cAMP/Epac but not of Rap1. Challenging sperm with calcium or 8-pCPT-2′-O-Me-cAMP boosts the exchange of GDP for GTP on Rab3A. Recombinant Epac does not release GDP from Rab3A in vitro, suggesting that the Rab3A-GEF activation by cAMP/Epac in vivo is indirect. We propose that Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place and another that elicits intracellular calcium release.During fertilization in eutherian mammals, the spermatozoon must penetrate the zona pellucida to reach the oolema. Only sperm that have completed the acrosome reaction (AR)⁴ can successfully accomplish this task (1). The AR is a regulated exocytosis where the membrane of the acrosome, the single dense core secretory granule in sperm, fuses to the plasma membrane surrounding the anterior portion of the head. This process releases hydrolytic enzymes stored in the granule. These enzymes, together with the physical thrust derived from strong flagellar beating, enable sperm to penetrate the zona pellucida (1, 2). Physiological agonists accomplish the AR by inducing an influx of calcium from the extracellular medium and the assembly of a conserved proteinaceous fusion machinery that includes Rab3A, α-SNAP/NSF, synaptotagmin, complexin, and neurotoxin-sensitive SNAREs; the AR also requires an efflux of calcium from inside the acrosome through IP₃-sensitive channels (reviewed in Refs. 3, 4).In certain neurons, neuroendocrine and exocrine acinar cells, cAMP potentiates calcium-dependent exocytosis. Either cAMP-dependent protein kinase (PKA) or the exchange protein directly activated by cAMP (Epac) can be the targets of cAMP in the cAMP-regulated exocytosis. On the other hand, cAMP is the principal trigger of regulated secretion in various non-neuronal cells (5–7). Likewise, an elevation of cAMP alone is sufficient to trigger exocytosis in human sperm. Moreover, calcium relies on endogenous cAMP to accomplish acrosomal release, and it does so through a PKA-insensitive pathway involving Epac. The stimulation of endogenous Epac by the selective cAMP analogue 8-(p-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) is sufficient to trigger the AR even in the absence of extracellular calcium. Furthermore, when Epac is sequestered with specific antibodies, cAMP, calcium (8), and recombinant Rab3A (this study) are unable to elicit exocytosis.Epac1 and Epac2 are multidomain proteins that consist of an N-terminal regulatory region and a C-terminal catalytic region (9–11). The regulatory domain harbors the cAMP-binding site, which auto-inhibits the catalytic activity in the absence of cAMP (12–15). The catalytic portion bears a guanine-nucleotide exchange factor (GEF) activity specific for Rap1 and Rap2 (16, 17). Like all small G proteins, Raps cycle between an inactive GDP-bound and an active GTP-bound conformation. The GDP-GTP cycle is regulated by GEFs that induce the release of the bound GDP to be replaced by the more abundant GTP and by GTPase-activating proteins that coax the intrinsic GTPase activity to rapidly hydrolyze bound GTP, returning the G proteins to the inactive GDP-bound state (18, 19). Most small G proteins are linked to biological membranes via lipid modifications at their C terminus; for instance, Rap2A is farnesylated, and Rap1A/B, Rap2B, and Rabs are geranylgeranylated (20, 21). Guanine nucleotide dissociation inhibitors (GDIs) remove Rabs from membranes by sequestration of their lipid tails (22).Extracellular stimuli often result in the activation of cellular adenylate cyclases and an increase in cAMP levels. By serving as a cAMP-binding protein with intrinsic GEF activity, Epac couples cAMP production to a variety of Rap-mediated processes such as the control of cell adhesion and cell-cell junction formation, water resorption, cell differentiation, inflammatory processes, etc. (9–11). Many are the effectors of Epac and Epac-Rap signaling. Of particular interest to us is the observation that Epac stimulates phospholipase Cϵ (PLCϵ) through the activation of Rap1 and -2, resulting in IP₃-mediated release of calcium from internal stores (23, 24). PLCϵ is an unusual enzyme with two catalytic activities as follows: the typical phosphatidylinositol 4,5-bisphosphate hydrolyzing PLC activity plus a Rap-GEF activity. Thus, PLCϵ acts both downstream and upstream of Ras-like GTPases, perhaps to guarantee sustained Rap signaling (25).During membrane fusion, Rab proteins direct the recognition and physical attachments of the compartments that are going to fuse (26, 27). This association, or tethering, represents one of the earliest known events in membrane fusion and is accomplished through the recruitment of tethering factors. Rab3A localizes to vesicles and secretory granules and is one of the isoforms directly implicated in regulated exocytosis of neurotransmitters and hormones (28). Rab3A interacts in a GTP-dependent manner with at least two effector proteins, rabphilin and Rim (29–31). Rab3A is present in the acrosomal region of human (32), rat (33), and mouse sperm (34). Rab3A (full-length recombinant protein or a synthetic peptide corresponding to the effector domain) stimulates human (32, 35) and ram (36) and inhibits rat sperm AR (33). Rab3A is required for the AR triggered by calcium (37, 38) and cAMP (8).Epac is a multifunctional protein in which cAMP exerts its effects not only by promoting the exchange of GDP for GTP on Rap but also by allosterically regulating other molecules (10). In exocytosis for instance, a number of Rap-independent, Epac-linked signaling pathways have been described. They include the interaction of Epac2 with Rim2 (39) and the Rim2-related protein Piccolo (40). Epac2 also stimulates exocytosis by interacting with SUR1 (41). Finally, Epac2 controls ryanodine-sensitive calcium channels that are involved in calcium-induced calcium release (CICR) from internal stores in insulin-secreting cells (42).In this study, we piece together the analysis of two phenomena as follows: calcium mobilization and protein-protein interactions preceding exocytosis. To the best of our knowledge, this constitutes the first integrated molecular model that includes both the assembly of the fusion and intravesicular calcium release protein machineries during regulated exocytosis. By enquiring further into the signaling pathways operating during sperm exocytosis, we have found more players than previously suspected, and we discovered that the key components of these cascades are not arranged in a linear sequence. Epac sits at a central point of the signaling cascade after which the exocytotic pathway splits into two limbs as follows: one that assembles the fusion machinery into place, and another that elicits the release of calcium from the acrosome; both need to act in concert to achieve exocytosis. Our results identify Rab3A for the first time as a downstream target for Epac and place this small GTPase as an early component of the “fusion machinery” branch of the pathway. They also show that Epac stimulates the exchange of GDP for GTP on Rap1 and that this protein, as well as a PLC, drives intracellular calcium mobilization. Finally, our data reveal that a soluble adenylyl cyclase (sAC) (43, 44) synthesizes the cAMP that activates Epac. Again, we believe that this is the first report linking sAC to an exocytotic event.