Small GTP-Binding Proteins of the Rho Family in the Dictyostelium Cytoskeleton
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Transcript of Small GTP-Binding Proteins of the Rho Family in the Dictyostelium Cytoskeleton
Protist, Vol. 149, 11-15, February 1998 © Gustav Fischer Verlag
PROTIST NEWS
Protist
Small GTP-Binding Proteins of the Rho Familyin the Dictyostelium Cytoskeleton
Dictyostelium discoideum has emerged as a widelyemployed model to investigate basic questions ofmolecular and cell biology (Maeda et al. 1997).Three features make D. discoideum an attractivemodel organism in which to stUGy the componentsof the actin cytoskeleton, the relationships amongthem and the elements involved in their regulation.First, Dictyostelium exhibits a particular life cycle inwhich free living amebae aggregate in response tochemotactic signals to build up a multicellular fruiting body in a process that requires the integrity ofthe actin cytoskeleton. Second, despite their apparent simplicity, Dictyostelium amebae are equippedwith a complex actin cytoskeleton that endows thecells with motile behavior comparable to that ofleukocytes. And third, Dictyostelium is amenable toa variety of biochemical, and molecular and cell biology techniques that make multidisciplinary approaches possible. In particular the ease of cultivation facilitates the isolation of proteins associatedwith the actin network; furthermore, the genome canbe easily manipulated by means of recombinantDNA techniques, the application which is vastlywidening our understanding of the role of cytoskeletal components.
Much is known about actin itself and about actinassociated proteins, their activities and subcellularlocalization, in particular in response to extra- andintracellular stimuli (Noegel and Luna 1995). Fromthese studies it is clear that regulatory componentsare needed that allow a cell to decide where andwhen the cytoskeleton has to be rearranged in response to a particular stimulus. Although a complete picture on the connections between signaltransduction pathways and reorganization of cytoskeletal structures is missing, evidence is accumulating on the role of small GTP-binding proteins ofthe Ras superfamily in this process.
Based on sequence homologies the Ras-relatedsmall GTPases can be grouped in to five families,each of them having a specific biological function:
Rab and ARF (as well as ARL or ARF-like proteins)are implicated in vesicle transport, Ran in nuclearprotein import, and Ras and Rho regulate signaltransduction pathways linking plasma membranereceptors to either growth and differentiation responses or to changes in the cytoskeletal organization respectively (Hall 1994). In this short review wewill focus on the members of the Rho family of GTPases described so far in Dictyostelium and willrefer to potential regulatory elements upstream aswell as downstream of these proteins that havebeen recognized in the last few years (Table 1).
Rho-Related GTPases and theCytoskeleton
Based on studies carried out primarily in mammaliancells, the Rho-related GTPases have been groupedinto three subfamilies, according to functional aspects. Rae proteins are involved in the formation oflamellipodia and membrane ruffles, Rho memberscoordinate stress fibre and adhesion plaque formation, and Cdc42 stimulates the formation of filopods(Hall 1994). Recent data indicate that besides regulation of the cytoskeleton organization, Rho-relatedGTPases are involved in a variety of other cell functions, including endocytosis, transcriptional regulation and growth control. Indeed, the pathways regulated by each of these GTPases are connected toeach other as well as to the pathways of other GTPases, especially those involving Ras (Ridley 1996).Not all processes dependent on Rho-like proteinsare present in Dictyostelium. These cells display anintense protrusive activity but do not have, for example, stress fibres and adhesion plaques, whichlargely simplifies studies on the role of Rho-like proteins.
Nine Rho-related proteins have been identified sofar in Dictyostelium using a variety of approaches.Bush et al. (1993) used degenerated oligodeoxy-
Protist, Vol. 149, 11-15, February 1998 © Gustav Fischer Verlag
PROTIST NEWS
Protist
Small GTP-Binding Proteins of the Rho Familyin the Dictyostelium Cytoskeleton
Dictyostelium discoideum has emerged as a widelyemployed model to investigate basic questions ofmolecular and cell biology (Maeda et al. 1997).Three features make D. discoideum an attractivemodel organism in which to stUGy the componentsof the actin cytoskeleton, the relationships amongthem and the elements involved in their regulation.First, Dictyostelium exhibits a particular life cycle inwhich free living amebae aggregate in response tochemotactic signals to build up a multicellular fruiting body in a process that requires the integrity ofthe actin cytoskeleton. Second, despite their apparent simplicity, Dictyostelium amebae are equippedwith a complex actin cytoskeleton that endows thecells with motile behavior comparable to that ofleukocytes. And third, Dictyostelium is amenable toa variety of biochemical, and molecular and cell biology techniques that make multidisciplinary approaches possible. In particular the ease of cultivation facilitates the isolation of proteins associatedwith the actin network; furthermore, the genome canbe easily manipulated by means of recombinantDNA techniques, the application which is vastlywidening our understanding of the role of cytoskeletal components.
Much is known about actin itself and about actinassociated proteins, their activities and subcellularlocalization, in particular in response to extra- andintracellular stimuli (Noegel and Luna 1995). Fromthese studies it is clear that regulatory componentsare needed that allow a cell to decide where andwhen the cytoskeleton has to be rearranged in response to a particular stimulus. Although a complete picture on the connections between signaltransduction pathways and reorganization of cytoskeletal structures is missing, evidence is accumulating on the role of small GTP-binding proteins ofthe Ras superfamily in this process.
Based on sequence homologies the Ras-relatedsmall GTPases can be grouped in to five families,each of them having a specific biological function:
Rab and ARF (as well as ARL or ARF-like proteins)are implicated in vesicle transport, Ran in nuclearprotein import, and Ras and Rho regulate signaltransduction pathways linking plasma membranereceptors to either growth and differentiation responses or to changes in the cytoskeletal organization respectively (Hall 1994). In this short review wewill focus on the members of the Rho family of GTPases described so far in Dictyostelium and willrefer to potential regulatory elements upstream aswell as downstream of these proteins that havebeen recognized in the last few years (Table 1).
Rho-Related GTPases and theCytoskeleton
Based on studies carried out primarily in mammaliancells, the Rho-related GTPases have been groupedinto three subfamilies, according to functional aspects. Rae proteins are involved in the formation oflamellipodia and membrane ruffles, Rho memberscoordinate stress fibre and adhesion plaque formation, and Cdc42 stimulates the formation of filopods(Hall 1994). Recent data indicate that besides regulation of the cytoskeleton organization, Rho-relatedGTPases are involved in a variety of other cell functions, including endocytosis, transcriptional regulation and growth control. Indeed, the pathways regulated by each of these GTPases are connected toeach other as well as to the pathways of other GTPases, especially those involving Ras (Ridley 1996).Not all processes dependent on Rho-like proteinsare present in Dictyostelium. These cells display anintense protrusive activity but do not have, for example, stress fibres and adhesion plaques, whichlargely simplifies studies on the role of Rho-like proteins.
Nine Rho-related proteins have been identified sofar in Dictyostelium using a variety of approaches.Bush et al. (1993) used degenerated oligodeoxy-
12 F. Rivero and A. A. Noegel
Table 1. Rho-related proteins, regulators and downstream and upstream elements involved in the organization ofthe actin cytoskeleton in Dictyostelium discoideum.
Protein
Rho-related proteinsRac1A,Rac1B,Rac1CRacARacBRacC
RacDRacERacF
RegulatorsDGAP1/DdRasGAP1
GAPADdRacGAPaimless RasGEF
Upstream elementsDdPIK1, DdPIK2,DdPIK3
Downstream elementsMIHCK
Subcellular localization
NDNDNDCortical enrichment; cytosol
NDCortical enrichment; cytosolCortical enrichment; cytosol
Cortical enrichment; cytosol
NDNDND
ND
ND
Null mutant phenotype
NANANANA
NACytokinesis defectNA
Aberrant development;cytokinesis defect(a)Cytokinesis defectNAChemotaxis defect
No defect in single mutantsddpik1- and ddpik2-.Defects in endocytosis andactin cytoskeleton organization in double mutantsddpik1-lddpik2-
NA
Reference
Bush et al. 1993Bush et al. 1993Bush et al. 1993Bush et al. 1993;Larochelle et al. 1997Bush et al. 1993Larochelle et al. 1996; 1997Rivero et aI., unpublished
Faix and Dittrich 1996;Lee et al. 1997Adachi et al. 1997Ludbrook et al. 1997Insall et al. 1996
Zhou et al. 1995;Buczynski et al. 1997
Lee et al. 1996
a. Phenotypes described by Faix and Dittrich (1996) and by Lee et al. (1997) are not consistent (see text)ND, not determined. NA, not available
nucleotide probes corresponding to conserved domains of the GTPases and isolated seven rho-related genes: rac1A, rac1B, rac1C and racA to racO.racE has been identified as the gene disrupted in acytokinesis mutant generated by REMI (restrictionenzyme mediated integration) (Larochelle et al.1996). Finally, we have used a PCR approach utilizing degenerated primers corresponding to twohighly conserved GTP-binding sites to isolate RacF(Rivero et aI., unpublished). Based on their high degree of primary sequence homology to mammalianRac proteins, all the Rho-related GTPases described so far for Oictyostelium have being designated as Rae, although functional studies are necessary to confirn this categorization.
Each of these rae genes displays a unique patternof expression, suggesting specific roles during thedifferent stages of Oictyostelium development. racBand race, for example, are expressed predominantly during vegetative growth and early develop-
ment, and racF is constitutively expressed throughout the entire life cycle. Except for RacE, the dataavailable to date are insufficient to assign a functional role for each of the Oictyostelium Rac proteins. Larochelle et al. (1996, 1997) have shown thatRacE is essential for cytokinesis, racE null mutantsbeing normal in other functions tested, like phagocytosis, chemotaxis and development. RacE appears to be located at the plasma membrane, sincea fusion protein of green fluorescence protein (GFP)with RacE localizes to the plasma membranethroughout the entire cell cycle, indicating that it isnot involved in the placement or timing of the contractile ring. A similar pattern of cortical distributionwas observed with GFP fusions of RacC (Larochelleet al. 1997) and RacF (Rivero et aI., unpublished)and can be related to structural features in the C-terminal part of the molecule, namely a prenylationmotif and a polybasic stretch. Since both elementsare present in all known Oictyostelium Racs (with the
12 F. Rivero and A. A. Noegel
Table 1. Rho-related proteins, regulators and downstream and upstream elements involved in the organization ofthe actin cytoskeleton in Dictyostelium discoideum.
Protein
Rho-related proteinsRac1A,Rac1B,Rac1CRacARacBRacC
RacDRacERacF
RegulatorsDGAP1/DdRasGAP1
GAPADdRacGAPaimless RasGEF
Upstream elementsDdPIK1, DdPIK2,DdPIK3
Downstream elementsMIHCK
Subcellular localization
NDNDNDCortical enrichment; cytosol
NDCortical enrichment; cytosolCortical enrichment; cytosol
Cortical enrichment; cytosol
NDNDND
ND
ND
Null mutant phenotype
NANANANA
NACytokinesis defectNA
Aberrant development;cytokinesis defect(a)Cytokinesis defectNAChemotaxis defect
No defect in single mutantsddpik1- and ddpik2-.Defects in endocytosis andactin cytoskeleton organization in double mutantsddpik1-lddpik2-
NA
Reference
Bush et al. 1993Bush et al. 1993Bush et al. 1993Bush et al. 1993;Larochelle et al. 1997Bush et al. 1993Larochelle et al. 1996; 1997Rivero et aI., unpublished
Faix and Dittrich 1996;Lee et al. 1997Adachi et al. 1997Ludbrook et al. 1997Insall et al. 1996
Zhou et al. 1995;Buczynski et al. 1997
Lee et al. 1996
a. Phenotypes described by Faix and Dittrich (1996) and by Lee et al. (1997) are not consistent (see text)ND, not determined. NA, not available
nucleotide probes corresponding to conserved domains of the GTPases and isolated seven rho-related genes: rac1A, rac1B, rac1C and racA to racO.racE has been identified as the gene disrupted in acytokinesis mutant generated by REMI (restrictionenzyme mediated integration) (Larochelle et al.1996). Finally, we have used a PCR approach utilizing degenerated primers corresponding to twohighly conserved GTP-binding sites to isolate RacF(Rivero et aI., unpublished). Based on their high degree of primary sequence homology to mammalianRac proteins, all the Rho-related GTPases described so far for Oictyostelium have being designated as Rae, although functional studies are necessary to confirn this categorization.
Each of these rae genes displays a unique patternof expression, suggesting specific roles during thedifferent stages of Oictyostelium development. racBand race, for example, are expressed predominantly during vegetative growth and early develop-
ment, and racF is constitutively expressed throughout the entire life cycle. Except for RacE, the dataavailable to date are insufficient to assign a functional role for each of the Oictyostelium Rac proteins. Larochelle et al. (1996, 1997) have shown thatRacE is essential for cytokinesis, racE null mutantsbeing normal in other functions tested, like phagocytosis, chemotaxis and development. RacE appears to be located at the plasma membrane, sincea fusion protein of green fluorescence protein (GFP)with RacE localizes to the plasma membranethroughout the entire cell cycle, indicating that it isnot involved in the placement or timing of the contractile ring. A similar pattern of cortical distributionwas observed with GFP fusions of RacC (Larochelleet al. 1997) and RacF (Rivero et aI., unpublished)and can be related to structural features in the C-terminal part of the molecule, namely a prenylationmotif and a polybasic stretch. Since both elementsare present in all known Oictyostelium Racs (with the
exception of RacA and RacD for which full-lengthsequences are not available), a similar pattern ofsubcellular localization is expected for all of them.
GAPs and GEFs, Regulators ofGTPase Activity
The GTPases can be considered as molecularswitches, being active as a GTP-bound form and inactive as a GDP-bound form. GTP-activating proteins (GAPs) enhance the hydrolysis of bound GTP,leading to inactivation of the small GTPases. Theopposite effect is achieved by guanine nucleotideexchange factors (GEFs).
The first GAP identified in Dictyostelium, DGAP1/DdRasGAP1, has been independently reported bytwo groups (Faix and Dittrich 1996; Lee at al. 1997),using different approaches. Faix and Dittrich (1996)screened an expression library using a monoclonalantibody raised against a fraction of actin-associated proteins, whereas Lee et al. (1997) used theyeast two-hybrid system with a constitutively activated mammalian Ras as bait. DGAP1/ DdRasGAP1possesses a catalytic RasGAP-related domain(GRD) very closely related to that of S. pombe Sar1and human IQGAP1 and acts in vitro on Dictyostelium RasD (Lee et al. 1997). Immunofluorescencestaining indicates a cytosolic localization and astrong enrichment in the cell cortex, and Westernand Northern blot analyses are consistent with apattern of expression during vegetative growth andearly development (Faix and Dittrich 1996). Theanalysis performed by both laboratories using mutant cells lacking this GAP yielded different and evencontradictory results. Faix and Dittrich (1996) foundan increased rate of growth on bacterial lawns andan aberrant pattern of development with multitipped aggregates and occasionally atypical fruitingbodies. Lee et al. (1997), on the other hand, reportedthat ddrasgap1 null cells are multinucleate in suspension, a defect counterbalanced by traction-mediated cytofission when cells are plated on a solidsubstratum; additionally, these cells develop abnormally after the mid-slug stage, ending with the formation of multiply branched structures unable toculminate and form spores. Interestingly, Faix andDittrich (1996) found a cytokinesis defect in cellsoverexpressing DGAP1 similar to the one describedby Lee et al. (1997) in their null mutant. A definitiveexplanation for these opposing results is lacking atpresent, but they could be attributed to differencesin the strains used for these studies.
A cytokinesis defect has also been reported incells lacking GAPA, a protein closely related to
Rho-Like GTPases in Dictyostelium 13
DGAP1/DdRasGAP1, particularly in its catalyticGRD (Adachi et al. 1997). GAPA was identified in ascreen of cytokinesis deficient mutants generatedby REMI. gapA null cells are giant and multinucleated when grown both in suspension and on a solidsubstratum, and the defect has been traced to latersteps of cell division, when the cytoplasmic bridgeconnecting the daughter cells severs. Interestingly,gapA null cells are able to develop and form normalfruiting bodies with viable spores.
The deficiencies in cytokinesis and/or developmental pattern apparent in all these mutants clearlypoint to an implication in the control of cytoskeletondependent processes. Mammalian IQGAPs do notinteract with Ras, but inhibit the actiVity of Cdc42and Rae (Tapon and Hall 1997). Sequence analysesindicate that this could also be the case for bothDGAP1/DdRasGAP1 and GAPA, in particulary, theinvariant motif essential for catalytic activity of theGRD for both Dictyostelium GAPs is more related tothat of IQGAP1 than to the one present in other RasGAP related proteins. Although biochemical studiesare needed to confirm this point, it cannot be excluded that the RasGAP-related proteins identifiedin Dictyostelium participate to different extents bothin Ras and Rae-dependent pathways. Therefore itwould be interesting to investigate the possible relationship between these GAPs and RacE, the onlyDictyostelium Rae for which a requirement in cytokinesis has been reported to date.
Mammalian RhoGAPs are characterized by aconserved region of sequence homology, namelythe RhoGAP domain, which is approximately 140amino acids in length (Lamarche and Hall 1994).This feature has been exploited by Ludbrook et al.(1997) to clone ddracgap using a PCR approach.ddracgap encodes for a 150 kDa RhoGAP homologin Dictyostelium. The RhoGAP domain of this protein which is situated at the N-terminus and is followed by a SH3 domain and a DH-PH combinationshows 20-25% identity to other RhoGAPs. TheDH-PH combination is present in all RhoGEFs, andwhether this domain is active as an exchange factorin DdRacGAP remains to be investigated. Suchparadoxical juxtaposition of functionally contrarydomains has been described also for two otherGAPs, Bcr and Abr (Lamarche and Hall 1994), andcan be reconciled in a double switch model in whichboth activities are exerted on elements regulatingtwo pathways, inhibiting one and activating theother. In vitro, DdRacGAP is active on DictyosteliumRac1 A and RacC, as well as on human RhoA andRac1, but not on human Ras (Ludbrook et al. 1997).Further biochemical studies and the generation of aknock-out mutant will help to identify the targets of
exception of RacA and RacD for which full-lengthsequences are not available), a similar pattern ofsubcellular localization is expected for all of them.
GAPs and GEFs, Regulators ofGTPase Activity
The GTPases can be considered as molecularswitches, being active as a GTP-bound form and inactive as a GDP-bound form. GTP-activating proteins (GAPs) enhance the hydrolysis of bound GTP,leading to inactivation of the small GTPases. Theopposite effect is achieved by guanine nucleotideexchange factors (GEFs).
The first GAP identified in Dictyostelium, DGAP1/DdRasGAP1, has been independently reported bytwo groups (Faix and Dittrich 1996; Lee at al. 1997),using different approaches. Faix and Dittrich (1996)screened an expression library using a monoclonalantibody raised against a fraction of actin-associated proteins, whereas Lee et al. (1997) used theyeast two-hybrid system with a constitutively activated mammalian Ras as bait. DGAP1/ DdRasGAP1possesses a catalytic RasGAP-related domain(GRD) very closely related to that of S. pombe Sar1and human IQGAP1 and acts in vitro on Dictyostelium RasD (Lee et al. 1997). Immunofluorescencestaining indicates a cytosolic localization and astrong enrichment in the cell cortex, and Westernand Northern blot analyses are consistent with apattern of expression during vegetative growth andearly development (Faix and Dittrich 1996). Theanalysis performed by both laboratories using mutant cells lacking this GAP yielded different and evencontradictory results. Faix and Dittrich (1996) foundan increased rate of growth on bacterial lawns andan aberrant pattern of development with multitipped aggregates and occasionally atypical fruitingbodies. Lee et al. (1997), on the other hand, reportedthat ddrasgap1 null cells are multinucleate in suspension, a defect counterbalanced by traction-mediated cytofission when cells are plated on a solidsubstratum; additionally, these cells develop abnormally after the mid-slug stage, ending with the formation of multiply branched structures unable toculminate and form spores. Interestingly, Faix andDittrich (1996) found a cytokinesis defect in cellsoverexpressing DGAP1 similar to the one describedby Lee et al. (1997) in their null mutant. A definitiveexplanation for these opposing results is lacking atpresent, but they could be attributed to differencesin the strains used for these studies.
A cytokinesis defect has also been reported incells lacking GAPA, a protein closely related to
Rho-Like GTPases in Dictyostelium 13
DGAP1/DdRasGAP1, particularly in its catalyticGRD (Adachi et al. 1997). GAPA was identified in ascreen of cytokinesis deficient mutants generatedby REMI. gapA null cells are giant and multinucleated when grown both in suspension and on a solidsubstratum, and the defect has been traced to latersteps of cell division, when the cytoplasmic bridgeconnecting the daughter cells severs. Interestingly,gapA null cells are able to develop and form normalfruiting bodies with viable spores.
The deficiencies in cytokinesis and/or developmental pattern apparent in all these mutants clearlypoint to an implication in the control of cytoskeletondependent processes. Mammalian IQGAPs do notinteract with Ras, but inhibit the actiVity of Cdc42and Rae (Tapon and Hall 1997). Sequence analysesindicate that this could also be the case for bothDGAP1/DdRasGAP1 and GAPA, in particulary, theinvariant motif essential for catalytic activity of theGRD for both Dictyostelium GAPs is more related tothat of IQGAP1 than to the one present in other RasGAP related proteins. Although biochemical studiesare needed to confirm this point, it cannot be excluded that the RasGAP-related proteins identifiedin Dictyostelium participate to different extents bothin Ras and Rae-dependent pathways. Therefore itwould be interesting to investigate the possible relationship between these GAPs and RacE, the onlyDictyostelium Rae for which a requirement in cytokinesis has been reported to date.
Mammalian RhoGAPs are characterized by aconserved region of sequence homology, namelythe RhoGAP domain, which is approximately 140amino acids in length (Lamarche and Hall 1994).This feature has been exploited by Ludbrook et al.(1997) to clone ddracgap using a PCR approach.ddracgap encodes for a 150 kDa RhoGAP homologin Dictyostelium. The RhoGAP domain of this protein which is situated at the N-terminus and is followed by a SH3 domain and a DH-PH combinationshows 20-25% identity to other RhoGAPs. TheDH-PH combination is present in all RhoGEFs, andwhether this domain is active as an exchange factorin DdRacGAP remains to be investigated. Suchparadoxical juxtaposition of functionally contrarydomains has been described also for two otherGAPs, Bcr and Abr (Lamarche and Hall 1994), andcan be reconciled in a double switch model in whichboth activities are exerted on elements regulatingtwo pathways, inhibiting one and activating theother. In vitro, DdRacGAP is active on DictyosteliumRac1 A and RacC, as well as on human RhoA andRac1, but not on human Ras (Ludbrook et al. 1997).Further biochemical studies and the generation of aknock-out mutant will help to identify the targets of
14 F. Rivero and A. A. Noegel
both domains of GAP and GEF, and to establish thefunction of DdRacGAP.
One RasGEF, the product of the aimless or aleAgene, has been described so far in Dictyostelium (Insail et al. 1996). Aimless was found during a screenfor aggregation-deficient mutants obtained usingREMI, and shows strong homology to diverse RasGEFs, in particular the Drosophila Sos and humanhSos1. Contrary to what was expected upon disruption of a Ras-controlled pathway, growth of aleA nullcells was not impaired. Biochemical analyses of thismutant indicate that the product of aimless is required for activation of adenylyl cyclase by G proteins and for chemotaxis to cAMP. Since aleA nullcells display diminished levels of F actin after stimulation with cAMP, it is tempting to speculate that aparticular subset of GTPase regulators links the Raspathway to cytoskeleton dependent activities likemotility in response to a chemoattractant.
Upstream and Downstream Elements
Substantial progress has been made during the lastyears in the identification of upstream and downstream elements connecting the initial receptorswith the Rho family members and these with thefinal cytoskeletal targets in a variety of systems (Ridley 1996; Tapon and Hall 1997).
Phosphatidylinositide 3-kinases (PI3K), a family ofenzymes involved in signal transduction and vesicletrafficking, have been implicated in the regulation ofthe actin cytoskeleton through activation of Rae GTPases. PI 3-kinases are heterodimers composed ofan 85 kDa regulatory subunit and a 110 kOa catalytic subunit. The 85 kDa subunit contains aRhoGAP domain whose role remains unclear, sinceit does not seem to display activity in vitro (Lamarche and Hall 1994). Three PI 3-kinases related tothe mammalian 110 kDa subunit have been identified in Dictyostelium using a molecular genetic approach and knockout mutants are available for twoof them, DdPIK1 and DdPIK2 (Zhou et al. 1995).While single mutants show no observable phenotype, double mutants are defective in pinocytosisand lysosome to postlysosome transport, display alterations in actin distribution and in the chemotacticresponse and develop abnormally (Zhou et al 1995;Buczynski et al. 1997). To what extent and throughwhich mechanisms these alterations are related withthe Rae pathway remains to be determined.
A recently described myosin I heavy chain kinase(MIHCK) provides a direct link between the Rae signaling pathway and myosin-dependent motile processes in Dictyostelium (Lee et al. 1996). MIHCK is a
98 kDa protein closely related to yeast Ste20p andmammalian PAK. These proteins are composed of aC-terminal protein kinase catalytic domain and ashort central region containing a Cdc42/Rac-binding motif common to other known targets of Raeand Cdc42. Indeed MIHCK has been shown to bindhuman Cdc42 and Rac1 , but not RhoA in an overlayassay. A model emerges in which MIHCK autophosphorylates in the presence of activated-Rae andsubsequently stimulates myosin I ATPase activity(as demonstrated for myosin IB and 10), contributingto pseudopod extension and endocytosis, a mechanism that parallels the Rho-induced stress fibre formation in fibroblasts through regulation of myosinphosphorylation.
Conclusion
Although we still have to deal with a fragmentarypicture, the gap between the membrane signaltransduction elements and the final cytoskeletal response begins to be filled. The diversity of methodologies that can be applied to Dictyostelium makethis organism a particularly well suited model inwhich to study the Rho/Rac/Cdc42 pathway. Thecoming years will see the discovery of new elementsof this pathway, but more work is still needed toidentify the targets of each of them. The generationof mutants of one or more of these elements combined with biochemical and cell biology studies willwithout doubt contribute to the establishment oftheir potential roles in the reorganization of the actincytoskeleton.
References
Adachi H, Takahashi Y, Hasebe T, Shirouzu M,Yokoyama S, Sutoh K (1997) Dictyostelium IQGAP-related protein specifically involved in the completion ofcytokinesis. J Cell Bioi 137: 891-898
Buczynski G, Grove B, Nomura A, Kleve M, Bush J,Firtel RA, Cardelli J (1997) Inactivation of two Dietyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis,lysosome to postlysosome transport, and actin cytoskeleton organization. J Cell Bioi 136: 1271-1286.
Bush J, Franek K, Cardelli J (1993) Cloning and characterization of seven novel Dictyostelium discoideumrae-related genes belonging to the rho family of GTPases. Gene 136: 61-68
Faix J, Dittrich W (1996) OGAP1, a homologue of rasGTPase activating proteins that controls growth, cyto-
14 F. Rivero and A. A. Noegel
both domains of GAP and GEF, and to establish thefunction of DdRacGAP.
One RasGEF, the product of the aimless or aleAgene, has been described so far in Dictyostelium (Insail et al. 1996). Aimless was found during a screenfor aggregation-deficient mutants obtained usingREMI, and shows strong homology to diverse RasGEFs, in particular the Drosophila Sos and humanhSos1. Contrary to what was expected upon disruption of a Ras-controlled pathway, growth of aleA nullcells was not impaired. Biochemical analyses of thismutant indicate that the product of aimless is required for activation of adenylyl cyclase by G proteins and for chemotaxis to cAMP. Since aleA nullcells display diminished levels of F actin after stimulation with cAMP, it is tempting to speculate that aparticular subset of GTPase regulators links the Raspathway to cytoskeleton dependent activities likemotility in response to a chemoattractant.
Upstream and Downstream Elements
Substantial progress has been made during the lastyears in the identification of upstream and downstream elements connecting the initial receptorswith the Rho family members and these with thefinal cytoskeletal targets in a variety of systems (Ridley 1996; Tapon and Hall 1997).
Phosphatidylinositide 3-kinases (PI3K), a family ofenzymes involved in signal transduction and vesicletrafficking, have been implicated in the regulation ofthe actin cytoskeleton through activation of Rae GTPases. PI 3-kinases are heterodimers composed ofan 85 kDa regulatory subunit and a 110 kOa catalytic subunit. The 85 kDa subunit contains aRhoGAP domain whose role remains unclear, sinceit does not seem to display activity in vitro (Lamarche and Hall 1994). Three PI 3-kinases related tothe mammalian 110 kDa subunit have been identified in Dictyostelium using a molecular genetic approach and knockout mutants are available for twoof them, DdPIK1 and DdPIK2 (Zhou et al. 1995).While single mutants show no observable phenotype, double mutants are defective in pinocytosisand lysosome to postlysosome transport, display alterations in actin distribution and in the chemotacticresponse and develop abnormally (Zhou et al 1995;Buczynski et al. 1997). To what extent and throughwhich mechanisms these alterations are related withthe Rae pathway remains to be determined.
A recently described myosin I heavy chain kinase(MIHCK) provides a direct link between the Rae signaling pathway and myosin-dependent motile processes in Dictyostelium (Lee et al. 1996). MIHCK is a
98 kDa protein closely related to yeast Ste20p andmammalian PAK. These proteins are composed of aC-terminal protein kinase catalytic domain and ashort central region containing a Cdc42/Rac-binding motif common to other known targets of Raeand Cdc42. Indeed MIHCK has been shown to bindhuman Cdc42 and Rac1 , but not RhoA in an overlayassay. A model emerges in which MIHCK autophosphorylates in the presence of activated-Rae andsubsequently stimulates myosin I ATPase activity(as demonstrated for myosin IB and 10), contributingto pseudopod extension and endocytosis, a mechanism that parallels the Rho-induced stress fibre formation in fibroblasts through regulation of myosinphosphorylation.
Conclusion
Although we still have to deal with a fragmentarypicture, the gap between the membrane signaltransduction elements and the final cytoskeletal response begins to be filled. The diversity of methodologies that can be applied to Dictyostelium makethis organism a particularly well suited model inwhich to study the Rho/Rac/Cdc42 pathway. Thecoming years will see the discovery of new elementsof this pathway, but more work is still needed toidentify the targets of each of them. The generationof mutants of one or more of these elements combined with biochemical and cell biology studies willwithout doubt contribute to the establishment oftheir potential roles in the reorganization of the actincytoskeleton.
References
Adachi H, Takahashi Y, Hasebe T, Shirouzu M,Yokoyama S, Sutoh K (1997) Dictyostelium IQGAP-related protein specifically involved in the completion ofcytokinesis. J Cell Bioi 137: 891-898
Buczynski G, Grove B, Nomura A, Kleve M, Bush J,Firtel RA, Cardelli J (1997) Inactivation of two Dietyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis,lysosome to postlysosome transport, and actin cytoskeleton organization. J Cell Bioi 136: 1271-1286.
Bush J, Franek K, Cardelli J (1993) Cloning and characterization of seven novel Dictyostelium discoideumrae-related genes belonging to the rho family of GTPases. Gene 136: 61-68
Faix J, Dittrich W (1996) OGAP1, a homologue of rasGTPase activating proteins that controls growth, cyto-
kinesis, and development in Dietyostelium diseoideum.FEBS Lett 394: 251-257
Hall A (1994) Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Bioi 10:31-54
Insall RH, Borleis J, Devreotes PN (1996) The aimlessRasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dietyostelium. Curr Bioi 6: 719-729
Lamarche N, Hall A (1994) GAPs for rho-related GTPases. Trends Genet 10: 436-440
Larochelle DA, Vithalani KK, De Lozanne A (1996) Anovel member of the rho family of small GTP-bindingprotein is specifically required for cytokinesis. J CellBioi 133: 1321-1329
Larochelle DA, Vithalani KK, De Lozanne A (1997)Role of the Dietyostelium racE in cytokinesis: Mutational analysis and localization studies by use of greenfluorescent protein. Mol Bioi Cell 8: 935-944
Lee S, Escalante R, Firtel RA (1997) A Ras GAP is essential for cytokinesis and spatial patterning in Dietyostelium. Development 124: 983-996
Lee SF, Egelhoff TT, Mahasneh A, Cote GP (1996)Cloning and characterization of a Dietyostelium myosinI heavy chain kinase activated by cdc42 and rac. J BioiChem 271: 27044-27048
Ludbrook SB, Eccleston JF, Strom M (1997) Cloningand characterization of a rhoGAP homolog from Dietyostelium diseoideum. J Bioi Chem 272: 15682-15686
Rho-Like GTPases in Dictyostelium 15
Maeda Y, Inouye K, Takeuchi I, eds (1997) Dietyostelium. A model system for cell and developmental biology. Universal Academy Press, Inc. Tokyo
Noegel AA, Luna E (1995) The Dietyostelium cytoskeleton. Experientia 51: 1135-1143
Ridley AJ (1996) Rho: theme and variations. Curr Bioi 6:1256-1264
Tapon N, Hall A (1997) Rho, Rac and Cdc42 GTPasesregulate the organization of the actin cytDskeleton. CurrOp Cell Bioi 9: 86-92
Zhou K, Takegawa K, Emr S, Firtel R (1995) A phosphatidylinositol (PI) kinase gene family in Dietyosteliumdiseoideum. Biological roles of putative mammalianp110 and yeast Vps34p PI 3-kinase homologs duringgrowth and development. Mol Cell Bioi 15: 5645-5656
Francisco Rivero1 and Angelika A. Noegel
Institut fOr Biochemie I, Medizinische Fakultat,Universitat zu K61n, Joseph-Stelzmann-Strasse 52,
D - 50931 K61n, Germany
1 Corresponding author;fax: 49-221 4786979
e-mail: [email protected]
kinesis, and development in Dietyostelium diseoideum.FEBS Lett 394: 251-257
Hall A (1994) Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Bioi 10:31-54
Insall RH, Borleis J, Devreotes PN (1996) The aimlessRasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dietyostelium. Curr Bioi 6: 719-729
Lamarche N, Hall A (1994) GAPs for rho-related GTPases. Trends Genet 10: 436-440
Larochelle DA, Vithalani KK, De Lozanne A (1996) Anovel member of the rho family of small GTP-bindingprotein is specifically required for cytokinesis. J CellBioi 133: 1321-1329
Larochelle DA, Vithalani KK, De Lozanne A (1997)Role of the Dietyostelium racE in cytokinesis: Mutational analysis and localization studies by use of greenfluorescent protein. Mol Bioi Cell 8: 935-944
Lee S, Escalante R, Firtel RA (1997) A Ras GAP is essential for cytokinesis and spatial patterning in Dietyostelium. Development 124: 983-996
Lee SF, Egelhoff TT, Mahasneh A, Cote GP (1996)Cloning and characterization of a Dietyostelium myosinI heavy chain kinase activated by cdc42 and rac. J BioiChem 271: 27044-27048
Ludbrook SB, Eccleston JF, Strom M (1997) Cloningand characterization of a rhoGAP homolog from Dietyostelium diseoideum. J Bioi Chem 272: 15682-15686
Rho-Like GTPases in Dictyostelium 15
Maeda Y, Inouye K, Takeuchi I, eds (1997) Dietyostelium. A model system for cell and developmental biology. Universal Academy Press, Inc. Tokyo
Noegel AA, Luna E (1995) The Dietyostelium cytoskeleton. Experientia 51: 1135-1143
Ridley AJ (1996) Rho: theme and variations. Curr Bioi 6:1256-1264
Tapon N, Hall A (1997) Rho, Rac and Cdc42 GTPasesregulate the organization of the actin cytDskeleton. CurrOp Cell Bioi 9: 86-92
Zhou K, Takegawa K, Emr S, Firtel R (1995) A phosphatidylinositol (PI) kinase gene family in Dietyosteliumdiseoideum. Biological roles of putative mammalianp110 and yeast Vps34p PI 3-kinase homologs duringgrowth and development. Mol Cell Bioi 15: 5645-5656
Francisco Rivero1 and Angelika A. Noegel
Institut fOr Biochemie I, Medizinische Fakultat,Universitat zu K61n, Joseph-Stelzmann-Strasse 52,
D - 50931 K61n, Germany
1 Corresponding author;fax: 49-221 4786979
e-mail: [email protected]