AbstractA new study shows that Ras2 regulates GPI-anchor synthesis in the ER. Reciprocally, the targeted enzyme GPI-GlcNAc transferase regulates Ras2 signal output. This novel intersection of Ras2 signaling and an ER-localized protein complex has interesting implications for Ras function

Hancock, John F

Elsevier - Publisher Connector 

GPI-Anchor Synthesis Ras Takes Charge

Previews
743
whether E2F3 is a repressor of Arf also in other cell these proapoptotic genes is growth regulated peaking
types and other species. Notwithstanding these open at or near the G1/S transition. This implies that mecha-
questions, the identification of E2F3 as a pivotal repres- nisms acting downstream to their transcriptional activa-
sor of Arf is an important addition to our understanding tion may play a critical role in inhibition of E2F-induced
of how E2Fs control cell proliferation and apoptosis in apoptosis during normal growth. Unraveling these mo-
normal and transformed cells. lecular mechanism(s) remains a challenging problem.
The data presented by Aslanian et al. also imply that,
similarly to the INK4a/Arf locus, the E2F3 locus affects
the activity of the pRB/E2F and ARF/p53 pathways. Doron Ginsberg
However, while the products of the INK4a/Arf locus Department of Molecular Cell Biology
upregulate the activities of pRB and p53, the products The Weizmann Institute of Science
of the E2F3 locus, E2F3a and E2F3b, antagonize pRB Rehovot 76100
and ARF/p53 activities, respectively. This raises the in- Israel
teresting possibility that under some settings amplifica-
tion of the E2F3 locus may contribute to tumor develop- Selected Reading
ment, similarly to inactivation of the INK4a/Arf locus that
is observed in many human cancers (Ruas and Peters, Aslanian, A., Iaquinta, P.J., Verona, R., and Lees, J.A. (2004). Genes
Dev. 18, 1413–1422.1998). Indeed, amplification of the E2F3 locus was docu-
mented in some human bladder tumors. It remains to Bates, S., Phillips, A.C., Clark, P.A., Stott, F., Peters, G., Ludwig,
be determined whether such amplification exists also R.L., and Vousden, K.H. (1998). Nature 395, 124–125.
in other tumors. DeGregori, J. (2002). Biochim. Biophys. Acta 1602, 131–150.
E2F1 can induce both proliferation and apoptosis, and Ginsberg, D. (2002). FEBS Lett. 529, 122–125.
understanding how its proapoptotic activity is inhibited
Leone, G., Nuckolls, F., Ishida, S., Adams, M., Sears, R., Jakoi, L.,during normal proliferation is a challenging problem. Of Miron, A., and Nevins, J.R. (2000). Mol. Cell. Biol. 20, 3626–3632.
note, the E2F3-mediated repression of Arf most proba-
Lowe, S.W., and Sherr, C.J. (2003). Curr. Opin. Genet. Dev. 13, 77–83.bly does not fully account for this inhibition: First, the
Ruas, M., and Peters, G. (1998). Biochim. Biophys. Acta 1378, F115–role of ARF in E2F1-induced p53-dependent apoptosis
F177.is debatable. Second, E2F1 induces apoptosis also in
Sherr, C.J. (2001). Nat. Rev. Mol. Cell Biol. 2, 731–737.a p53-independent manner and this is attributed, at least
Sherr, C.J., and McCormick, F. (2002). Cancer Cell 2, 103–112.in part, to direct transcriptional activation of proapo-
ptotic genes including p73, Apaf-1, caspases, and BH3- Takahashi, Y., Rayman, J.B., and Dynlacht, B.D. (2000). Genes Dev.
14, 804–816.only proteins (Ginsberg, 2002). Unlike Arf, expression of
eri1 cells have an additional phenotype that cannotGPI-Anchor Synthesis:
be explained by increased Ras signaling: they grow nor-Ras Takes Charge mally at 23C but not at 37C. It is the investigation of
this ts (temperature-sensitive) phenotype that forms the
intriguing second part of the story published in the May
28 issue of Cell (Sobering et al., 2004). Levin and col-
A new study shows that Ras2 regulates GPI-anchor leagues show that eri1 cells are extremely sensitive to
synthesis in the ER. Reciprocally, the targeted enzyme lysis at the nonpermissive temperature, a phenotype
GPI-GlcNAc transferase regulates Ras2 signal output. that is rescued by increasing the osmotic pressure of
This novel intersection of Ras2 signaling and an ER- the growth media and therefore indicative of a cell wall
localized protein complex has interesting implications defect. A genetic screen for multicopy suppressors of ts
for Ras function. growth then turned up GFA1, an enzyme that catalyzes
production of glucosamine-6-phosphate—the rate-lim-
Eri1 (for endoplasmic reticulum-associated Ras inhibitor iting step for UDP-N-acetylglucosamine (UDP-GlcNAc)
1) was recently isolated in a genetic screen for yeast production. The conclusion that GFA1 drives up cellular
with growth defects that were additive with defective levels of glucosamine-6-phosphate to compensate for
protein kinase C: a regulator of cell wall biogenesis (So- loss of Eri1 was confirmed by showing a similar rescue
bering et al., 2003). Interestingly, eri1 yeast are heat of growth at 37C if eri1 cells were supplied with an
shock sensitive and grow invasively on agar, pheno- exogenous source of glucosamine.
types that indicate hyperactive Ras signaling. The heat UDP-GlcNAc is used for protein N-glycosylation, and
shock sensitivity of eri1 strains is milder than that in- the synthesis of chitin and GPI-anchors. Chitin and GPI-
duced by constitutively active Ras2Val19 but is sup- anchored proteins are, respectively, minor and major
pressed by deleting Ras2 or overexpressing Ira2 (a structural components of the yeast cell wall. Defects in
GTPase activating protein). Eri1 and Ras2 form a com- N-glycosylation and chitin synthesis were excluded by
plex when coexpressed in yeast, although immunofluo- showing normal maturation of N-glycosylated CPY and
rescence microscopy shows Eri1 is localized to the en- by the finding that chitin synthesis is actually increased,
partly because of an upregulation of endogenous GFA1doplasmic reticulum (ER).
Developmental Cell
744
in eri1 cells. There was also no increase in the severity Ras function in mammalian cells? Although Ras2 and
of the ts phenotype when chitin synthase was deleted. In mammalian Ras utilize different effector proteins, the
contrast, maturation of the GPI-anchored Gas1 protein Levin study will surely prompt investigation of whether
was severely retarded, with a concomitant failure to mammalian GPI-GlcNAc transferase is also regulated
transport Gas1 out of the ER. Biochemical analysis iden- by Ras. Mammalian Ras proteins transiently associate
tified the cause as failure of GPI-anchor synthesis. with the ER for posttranslational processing of the
The first step in the biosynthesis of GPI-anchors is C-terminal CAAX motif. Although it is possible that dur-
transfer of GlcNAc from UDP-GlcNAc to an acceptor ing biosynthetic transit GlcNAc transferase and Ras
phosphatidylinositol molecule, catalyzed by a multisub- could reciprocally inhibit each other’s activity, this pre-
unit ER-localized enzyme complex: GPI-GlcNAc trans- supposes that there is sufficient Ras present in the ER
ferase. Direct assay of this enzyme in eri1 cells re- at steady state to regulate GlcNAc transferase. To date,
vealed 2% residual activity, identifying the phenotype only ectopically expressed mutant forms of Ras appear
as a problem of GPI-anchor synthesis. Four proteins to stably associate with the ER (Chiu et al., 2002).
make up the yeast GPI-GlcNAc transferase, Gpi1, Gpi2, If the regulation of GPI-GlcNAc by Ras2 is indirect,
Gpi3, and Gpi15. The new data suggest that Eri1 may the hyperactive Ras2 phenotype in eri1 and gpi1 cells
be a fifth: for example, Gpi2 is present in Eri1 immuno- may reflect a change in the composition of the yeast cell
precipitates when the proteins are coexpressed, and, membrane that allows increased Ras2 signaling from the
analogous to the original results with Eri1, Ras2Val19 can plasma membrane rather than loss of a direct inhibitory
form a complex with Gpi2 if the Ras effector domain is interaction with GPI-GlcNAc. In mammalian cells, the
intact. Furthermore, deletion of Gpi1 faithfully replicates lateral segregation of Ras proteins to different plasma
the eri1 phenotype, including hyperactive Ras signal- membrane microdomains has a profound effect on sig-
ing. Ras2 also regulates UDP-GlcNAc activity: ectopic nal output. The nature of these domains is debated, but
expression of Ras2Val19 inhibits UDP-GlcNAc activity cholesterol, actin, and scaffold proteins are all important
to 5% wild-type, whereas in ras2 cells its activity is in their organization (Hancock, 2003). Mutations in GPI-
increased 8- to 11-fold. GlcNAc that block GPI-anchor synthesis cause substan-
The results show that GPI-GlcNAc is a novel Ras ef- tial increases in plasma membrane cholesterol (Abrami
fector, but one whose activity is negatively regulated by et al., 2001) that could influence Ras microlocalization
activated Ras. Simultaneously, GPI-GlcNAc negatively and signal output. Given that yeast also have ergosterol-
regulates Ras. The authors propose that Ras, in parallel, dependent (rather than cholesterol-dependent) lipid raft
inhibits GPI-GlcNAc and stimulates adenyl cyclase, the domains (Bagnat and Simons, 2002), these concepts
established Ras effector. They also propose that the from mammalian biology may be relevant.
hyperactive Ras phenotype in eri1 and gpi1 cells is
Finally, there is an interesting, albeit rare hematologi-
a consequence of losing an inhibitor of Ras signaling—
cal disorder, PNH, characterized by somatic mutations
GPI-GlcNAc transferase—thus in this model the hyper-
in the catalytic subunit of mammalian GlcNAc trans-
active Ras phenotype is mediated by adenyl cyclase.
ferase. GPI-deficient hematopoietic stem cells have aRas2 therefore regulates cell wall architecture by inhib-
growth advantage over normal GPI-anchor-expressingiting GPI-anchor protein synthesis and perhaps by in-
stem cells that allows for their clonal expansion (Inouecreasing chitin production. The resulting changes in cell
et al., 2003). The reason why is poorly understood andwall structure could be important in the change to fila-
the subject of much speculation; in light of the currentmentous/invasive growth.
paper, we can add the suggestion of increased basalThis crosstalk between Ras signaling, GPI-anchor
Ras signaling—this would at least explain the recentlysynthesis, and cell wall structure adds an exciting new
reported ubiquitous upregulation in PNH cells of EGR-1,perspective to Ras signal transduction. There are, how-
a MAPK-regulated transcription factor (Inoue et al.,ever, some as yet unanswered questions. For example,
2003). Whatever the molecular mechanisms in play,exactly what is the mechanism whereby Ras2 and GPI-
crosstalk between Ras signaling and GPI-anchor syn-GlcNAc are reciprocally regulated? The authors advo-
thesis opens a Pandora’s box of intriguing possibilitiescate that this occurs through a direct protein/protein
for yeast and mammalian cell biologists.interaction; the data show that complex formation be-
tween Eri1, Gpi2, and Ras2 is possible, although pre-
cisely which component(s) of GPI-GlcNAc directly con-
tacts Ras2 remains to be identified. Nevertheless, an John F. Hancock
important issue here is that Ras and GPI-GlcNAc are Institute for Molecular Bioscience
localized to nonoverlapping membranes (plasma mem- University of Queensland
brane and ER, respectively), and even in cells ectopically Brisbane 4072
expressing Eri1 and Ras2 less than 20% of the proteins Australia
colocalize on the ER (Sobering et al., 2003). It therefore
remains formally possible that the actual regulation of
Selected ReadingGPI-GlcNAc activity by Ras2 is downstream of adenyl
cyclase; if so, then GPI-GlcNAc activity in Ras2Val19 cells
Abrami, L., Fivaz, M., Kobayashi, T., Kinoshita, T., Parton, R.G., andshould be restored by high-copy expression of PDE2;
van der Goot, F.G. (2001). J. Biol. Chem. 276, 30729–30736.
a cAMP phosphodiesterase.
Bagnat, M., and Simons, K. (2002). Biol. Chem. 383, 1475–1480.What are the implications of this novel intersection of
Ras2 signaling and GPI-anchor production in the ER for Chiu, V.K., Bivona, T., Hach, A., Sajous, J.B., Silletti, J., Wiener, H.,
Previews
745
Johnson, R.L., 2nd, Cox, A.D., and Philips, M.R. (2002). Nat. Cell Sobering, A.K., Romeo, M.J., Vay, H.A., and Levin, D.E. (2003). Mol.
Cell. Biol. 23, 4983–4990.Biol. 4, 343–350.
Hancock, J.F. (2003). Nat. Rev. Mol. Cell Biol. 4, 373–384. Sobering, A.K., Watanabe, R., Romeo, M.J., Yan, B.C., Specht, C.A.,
Orlean, P., Riezman, H., and Levin, D.E. (2004). Cell 117, 637–648.Inoue, N., Murakami, Y., and Kinoshita, T. (2003). Int. J. Hematol.
77, 107–112.
these authors have shown that application of hydro-Putting the Squeeze
static pressure from the apical side of cells cultured onon Mechanotransduction filters stimulates a variety of events including metallo-
proteinase-dependent growth factor secretion and col-
lagen secretion by nearby mesenchymal cells, events
that may model airway thickening in asthma (Swartz et
al., 2001). They now report that the space between theBoth mechanical and chemical stimuli guide tissue
cells narrows in response to pressure and suggest thatfunction. In a recent paper, Tschumperlin et al. pro-
if the growth factor HB-EGF is secreted at a constantposed that pressure acting on airway epithelium elicits
rate into this compartment, narrowing will result in in-mechanotransduction not by directly altering bio-
creased HB-EGF concentration. They show that EGFchemical signaling but by regulating extracellular fluid
receptors are activated, that blocking these receptorsvolume to modulate ligand-receptor interactions.
inhibits some of the responses to pressure, and that
these events can be accurately predicted by a simple
Asthma is generally considered an inflammatory dis- first-order diffusion model for this process.
ease, often triggered by allergy or infection. It involves What is the evidence that such a mechanism exists
progressive airway remodeling to thicken the bronchial in vivo? Acetylcholine stimulation of canine bronchiolar
wall and hyperresponsiveness of airways to contractile smooth muscle results in intraluminal pressures of 30
stimuli. Remodeling contributes to decreased airflow cm H2O (Gunst and Stropp, 1988), corresponding to av-
during constriction and possibly to smooth muscle hy- erage circumferential stresses of 45 cm H2O (Ressler
perresponsiveness. Asthma research and treatment et al., 2000). Tschumperlin et al. confirmed that maximal
have focused largely on mechanisms of inflammation. bronchoconstriction with methacholine in the mouse
However, based on the notion that airway constriction trachea significantly increased EGFR phosphorylation,
is an intrinsically mechanical event, some investigators implicating the bronchial epithelium as a mechano-
have begun to explore the idea that remodeling could chemical transducer of intraluminal pressure. The valid-
be a consequence of airway constriction. Their work
ity of a pressure-induced decrease in lateral intercellular
has used a variety of model systems to show that me-
space (LIS) volume is less clear, since the expected
chanical stimulation of airway epithelium can trigger
mechanical environment differs among adjacent epithe-
many of the events involved in airway thickening, includ-
lial cells in the constricted airway wall. Smooth muscleing cell proliferation and deposition of a collagenous
contraction causes the epithelium to buckle and fold soextracellular matrix. This group has now published evi-
that regions of epithelial cells might be compresseddence for a novel mechanism for mechanotransduction
against each other along their apical surfaces (Figurethat radically departs from current thinking in this area.
1). Cells at the tops of the folds (facing the airway lumen)The ability of cells to convert mechanical forces into
are not likely to experience similar pressure increases,biochemical regulatory information is crucial for normal
and the potential effects of this heterogeneity in forcephysiology and pathological processes in many tissues.
distribution at the cell length scale is not addressed. OnExamples include bone adaptation to exercise or micro-
the other hand, these cells may experience increasedgravity and the strong link between locations of athero-
circumferential tension on tight junctions and compres-genesis and regional hemodynamics. Force or strain
sion of their basolateral surfaces due to the high invertedcan activate mechanosensitive ion channels at the cell
radius of curvature underlying the fold. Further work willsurface, alter the conformation of extracellular matrix
be required to determine whether these circumferentialproteins, or alter the function of tension-sensitive com-
stresses narrow the LIS in these regions and whetherponents of the cytoskeleton (Davies, 1995). Forces may
these regions correlate with remodeling. Interestingly,also alter physical properties of membrane microdo-
in mammary epithelium where milk accumulation at themains such as lipid rafts or caveolae to control assembly
end of nursing induces local epithelial cell apoptosis,of signaling complexes (Ferraro et al., 2004). Thus, it is
hydrostatic pressure has been proposed to mediate thisgenerally thought that cells contain force-sensing ele-
effect (Marti et al., 1997). The mechanism proposed byments and are capable of integrating a complex array
Tschumperlin et al. could be involved.of environmental cues.
If autocrine signaling within the LIS is functionally rele-A recent paper (Tschumperlin et al., 2004) provides
vant, then paracellular movement of fluid is likely toevidence for a distinct model for mechanotransduction.
influence the process. Tissues such as intestine andTschumperlin and coauthors study cultured airway epi-
glandular epithelium have regulated fluid flow throughthelium using application of hydrostatic pressure as a
model for mechanotransduction. Previous studies from the LIS that should affect ligand concentrations within


file:///data/remote/core/dit/data/elsevier/pdf/ee1/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMTUzNDU4MDcwNDAwMTczeA==.pdf

GPI-Anchor Synthesis Ras Takes Charge

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector