Protein prenylation: more than just glue?

As with other lipid modifications of proteins, prenylation now appears to be critically important in the regulation of protein function. Recent research has led to an explosion of information concerning prenylation signals, prenyl transferase enzymes and the role of prenylation in protein-membrane interactions. Experiments have examined the role of prenylation in protein function and the results suggest that protein prenylation may be involved in facilitating proper subcellular localization, promoting protein-protein and protein-membrane interactions and regulating protein function.

Structure and biological effects of lipid modifications on proteins.

Both the prevalence of lipid modifications of proteins and their importance for protein function and cellular localization have been widely observed. The advances made during the past year in defining the enzymology of lipid addition and in understanding the biological consequences of these modifications on protein function are discussed.

The Ras branch of small GTPases: Ras family members don't fall far from the tree.

The Ras branch of the Ras superfamily consists of small GTPases most closely related to Ras and include the R-Ras, Rap, Ral, Rheb, Rin and Rit proteins. Although our understanding of Ras signaling and biology is now considerable, recent observations suggest that Ras function is more complex than previously believed. First, the three Ras proteins may not be functionally identical. Second, Ras function involves functional cross-talk with their close relatives.

Role of DLC-1, a tumor suppressor protein with RhoGAP activity, in regulation of the cytoskeleton and cell motility.

DLC-1 was originally identified as a potential tumor suppressor. One of the key biochemical functions of DLC-1 is to serve as a GTPase activating protein (GAP) for members of the Rho family of GTPases, particularly Rho A-C and Cdc 42. Since these GTPases are critically involved in regulation of the cytoskeleton and cell migration, it seems clear that DLC-1 will also influence these processes. In this review we examine basic aspects of the actin cyoskeleton and how it relates to cell motility. We then delineate the characteristics of DLC-1 and other members of its family, and describe how they may have multiple effects on the regulation of cell polarity, actin organization, and cell migration.

The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling.

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

Understanding Ras: 'it ain't over 'til it's over'.

Since 1982, Ras has been the subject of intense research scrutiny, focused on determining the role of aberrant Ras function in human cancers and defining the mechanism by which Ras mediates its actions in normal and neoplastic cells. The long-term goal has been to develop antagonists of Ras as novel approaches for cancer treatment. Although impressive strides have been made in these endeavours, and our knowledge of Ras is quite extensive, it appears that we are at the beginning, rather than at the end, of fully understanding Ras function. This review highlights new issues that have further complicated our efforts to understand Ras.

Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia.

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens-type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E-cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine-phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras-transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.

R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism.

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.

Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain.

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.

Identity of HeLa cell determinants acquired by vesicular stomatitis virus with a tumor antigen.

Growth of vesicular stomatitis virus (VSV) in HeLa cells results in progeny containing non-VSV antigens with a molecular weight around 75,000. The non-VSV antigens were detected by antiserums to HeLa cell determinants. These antiserums precipitate whole virions but do not neutralize them. Because one of the antiserums is directed to a tumor-specific surface antigen of HeLa cells, it appears that VSV specifically acquires such antigens during its passage through human tumor cells.

Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal.

The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.

GTP binding by class II transactivator: role in nuclear import.

Class II transactivator (CIITA) is a global transcriptional coactivator of human leukocyte antigen-D (HLA-D) genes. CIITA contains motifs similar to guanosine triphosphate (GTP)-binding proteins. This report shows that CIITA binds GTP, and mutations in these motifs decrease its GTP-binding and transactivation activity. Substitution of these motifs with analogous sequences from Ras restores CIITA function. CIITA exhibits little GTPase activity, yet mutations in CIITA that confer GTPase activity reduce transcriptional activity. GTP binding by CIITA correlates with nuclear import. Thus, unlike other GTP-binding proteins, CIITA is involved in transcriptional activation that uses GTP binding to facilitate its own nuclear import.

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

Requirement of NF-kappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras.

The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-kappaB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-kappaB. These results provide an explanation for the requirement of NF-kappaB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.

Human cell hybrids: analysis of transformation and tumorigenicity.

Intraspecific human-human cell hybrids provide a stable model system with which to investigate the genetic control of transformed and tumorigenic phenotypes. Using this system it has been shown that these phenotypes are under separate genetic control. Furthermore, the tumorigenic phenotype can be complemented by fusion of different tumorigenic cells, resulting in nontumorigenic hybrids. This system also provides information on the control of differentiated function. Molecular cytogenetic techniques should reveal the nature of the chromosomal control of neoplastic transformation.

Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer.

Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation. Aberrant regulation of MAPK cascades contribute to cancer and other human diseases. In particular, the extracellular signal-regulated kinase (ERK) MAPK pathway has been the subject of intense research scrutiny leading to the development of pharmacologic inhibitors for the treatment of cancer. ERK is a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine/threonine kinases. Raf activates the MAPK/ERK kinase (MEK)1/2 dual-specificity protein kinases, which then activate ERK1/2. The mutational activation of Raf in human cancers supports the important role of this pathway in human oncogenesis. Additionally, the Raf-MEK-ERK pathway is a key downstream effector of the Ras small GTPase, the most frequently mutated oncogene in human cancers. Finally, Ras is a key downstream effector of the epidermal growth factor receptor (EGFR), which is mutationally activated and/or overexpressed in a wide variety of human cancers. ERK activation also promotes upregulated expression of EGFR ligands, promoting an autocrine growth loop critical for tumor growth. Thus, the EGFR-Ras-Raf-MEK-ERK signaling network has been the subject of intense research and pharmaceutical scrutiny to identify novel target-based approaches for cancer treatment. In this review, we summarize the current status of the different approaches and targets that are under evaluation and development for the therapeutic intervention of this key signaling pathway in human disease.

Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.

The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.

The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras.

We have used oligonucleotide-directed mutagenesis to replace the N-terminal amino acids of p21v-ras with residues which mimic the amino terminus of p60v-src. p21v-ras protein possessing only the first five amino acids of p60src was not myristylated, while substitution of residue 6 (serine) produced a protein p21(GSSKS) which incorporated [3H]myristic acid that was stable to hydroxylamine, sensitive to inhibitors of protein synthesis, and found in both the normally nonacylated precursor and mature forms of p21(GSSKS). This defines the minimum framework of the p60v-src myristylation signal (glycine 2 and serine 6) and identifies serine 6 as a crucial part of that signal for myristylation of a protein in vivo.