The contributions of the HRas C-terminus and its lipid modifications to HRas signal transduction events and membrane binding

In the GTP bound state, Ras proteins activate multiple downstream effector proteins, the combination of which regulate cell growth, differentiation, and changes in cell shape. However, Ras proteins must undergo C-terminal lipidation before they can support biological activity. Specifically, the C-terminus of the HRas protein must be modified by a farnesyl at Cys186 and two palmitates at Cys181 and Cys184. The current paradigm suggests that palmitate is not attached without prior farnesyl addition and that these post-translational lipid modifications are only necessary for membrane association. However, emerging evidence suggests that the C-terminus and its lipid modifications may also be responsible for Ras mediated signal transduction and may represent a previously unsuspected mechanism to control either normal or malignant cell growth;Our studies with two novel mutants of HRas, ExtRas and G43:Ras, demonstrated that palmitoylation could occur in the absence of farnesylation, and begin to explore the role of palmitoylation and the C-terminus in interactions with effector proteins and membrane association. Specifically, the efficiency of membrane association via palmitoylation signals alone, either at the N-terminus (G43:HRas) or the C-terminus (ExtRas), is significantly less than that of a farnesyl, palmitate combination. These two novel HRas proteins also possess different biological activities despite the fact that they contain the same core Ras effector sequence (residues 32--40) and flanking residues which are important for interactions with all identified downstream effector proteins. Further analysis of the unusual biological activity Ext61L protein has found an imbalance in the activation of two specific Ras effector proteins which may underlie its unusual biological activity. These studies emphasize that palmitate and isoprenoid modifications have distinct structural and biological roles and point toward an unexpected participation of the HRas C-terminus in signal transduction

Mutation of Ha-Ras C Terminus Changes Effector Pathway Utilization

In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions

A Non-farnesylated Ha-Ras Protein Can Be Palmitoylated and Trigger Potent Differentiation and Transformation

Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins

The contributions of the HRas C-terminus and its lipid modifications to HRas signal transduction events and membrane binding

In the GTP bound state, Ras proteins activate multiple downstream effector proteins, the combination of which regulate cell growth, differentiation, and changes in cell shape. However, Ras proteins must undergo C-terminal lipidation before they can support biological activity. Specifically, the C-terminus of the HRas protein must be modified by a farnesyl at Cys186 and two palmitates at Cys181 and Cys184. The current paradigm suggests that palmitate is not attached without prior farnesyl addition and that these post-translational lipid modifications are only necessary for membrane association. However, emerging evidence suggests that the C-terminus and its lipid modifications may also be responsible for Ras mediated signal transduction and may represent a previously unsuspected mechanism to control either normal or malignant cell growth;Our studies with two novel mutants of HRas, ExtRas and G43:Ras, demonstrated that palmitoylation could occur in the absence of farnesylation, and begin to explore the role of palmitoylation and the C-terminus in interactions with effector proteins and membrane association. Specifically, the efficiency of membrane association via palmitoylation signals alone, either at the N-terminus (G43:HRas) or the C-terminus (ExtRas), is significantly less than that of a farnesyl, palmitate combination. These two novel HRas proteins also possess different biological activities despite the fact that they contain the same core Ras effector sequence (residues 32--40) and flanking residues which are important for interactions with all identified downstream effector proteins. Further analysis of the unusual biological activity Ext61L protein has found an imbalance in the activation of two specific Ras effector proteins which may underlie its unusual biological activity. These studies emphasize that palmitate and isoprenoid modifications have distinct structural and biological roles and point toward an unexpected participation of the HRas C-terminus in signal transduction.</p

Mutation of Ha-Ras C Terminus Changes Effector Pathway Utilization

In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions.This research was originally published in The Journal of Biological Chemistry. Michelle A. Booden, Donald S. Sakaguchi, and Janice E. Buss. Mutation of Ha-Ras C Terminus Changes Effector Pathway Utilization. The Journal of Biological Chemistry. 2000, 275: 23559-23568. © the American Society for Biochemistry and Molecular Biology.</p

Critical but Distinct Roles for the Pleckstrin Homology and Cysteine-Rich Domains as Positive Modulators of Vav2 Signaling and Transformation

Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo

Leukemia-Associated Rho Guanine Nucleotide Exchange Factor Promotes Gαq-Coupled Activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein α subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of Gα12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific Gα subunits and couple Gα activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with Gα12 and Gα13 but also with Gαq. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of Gαq, Gα12, and Gα13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to Gαq and Gα13. Activated Gαq, as well as Gα12 and Gα13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three Gα subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways

Persistent Signaling by Dysregulated Thrombin Receptor Trafficking Promotes Breast Carcinoma Cell Invasion

Increased expression of protease-activated receptor 1 (PAR1), a G protein-coupled receptor for thrombin, has previously been correlated with breast carcinoma cell invasion. PAR1 is irreversibly proteolytically activated, internalized, and sorted directly to lysosomes, a critical process for the termination of signaling. We determined that activated PAR1 trafficking is severely altered in metastatic breast carcinoma cells but not in nonmetastatic or normal breast epithelial cells. Consequently, the proteolytically activated receptor is not sorted to lysosomes and degraded. Altered trafficking of proteolytically activated PAR1 caused sustained activation of phosphoinositide hydrolysis and extracellular signal-regulated kinase signaling, even after thrombin withdrawal, and enhanced cellular invasion. Thus, our results reveal that a novel alteration in trafficking of activated PAR1 causes persistent signaling and, in addition to other processes and proteins, contributes to breast carcinoma cell invasion