small gtp-binding proteins of the rho family in the dictyostelium cytoskeleton

5
Protist, Vol. 149, 11-15, February 1998 © Gustav Fischer Verlag PROTIST NEWS Protist Small GTP-Binding Proteins of the Rho Family in the Dictyostelium Cytoskeleton Dictyostelium discoideum has emerged as a widely employed model to investigate basic questions of molecular and cell biology (Maeda et al. 1997). Three features make D. discoideum an attractive model organism in which to stUGy the components of the actin cytoskeleton, the relationships among them and the elements involved in their regulation. First, Dictyostelium exhibits a particular life cycle in which free living amebae aggregate in response to chemotactic signals to build up a multicellular fruit- ing body in a process that requires the integrity of the actin cytoskeleton. Second, despite their appar- ent simplicity, Dictyostelium amebae are equipped with a complex actin cytoskeleton that endows the cells with motile behavior comparable to that of leukocytes. And third, Dictyostelium is amenable to a variety of biochemical, and molecular and cell biol- ogy techniques that make multidisciplinary ap- proaches possible. In particular the ease of cultiva- tion facilitates the isolation of proteins associated with the actin network; furthermore, the genome can be easily manipulated by means of recombinant DNA techniques, the application which is vastly widening our understanding of the role of cytoskele- tal components. Much is known about actin itself and about actin associated proteins, their activities and subcellular localization, in particular in response to extra- and intracellular stimuli (Noegel and Luna 1995). From these studies it is clear that regulatory components are needed that allow a cell to decide where and when the cytoskeleton has to be rearranged in re- sponse to a particular stimulus. Although a com- plete picture on the connections between signal transduction pathways and reorganization of cyto- skeletal structures is missing, evidence is accumu- lating on the role of small GTP-binding proteins of the Ras superfamily in this process. Based on sequence homologies the Ras-related small 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 nuclear protein import, and Ras and Rho regulate signal transduction pathways linking plasma membrane receptors to either growth and differentiation re- sponses or to changes in the cytoskeletal organiza- tion respectively (Hall 1994). In this short review we will focus on the members of the Rho family of GT- Pases described so far in Dictyostelium and will refer to potential regulatory elements upstream as well as downstream of these proteins that have been recognized in the last few years (Table 1). Rho-Related GTPases and the Cytoskeleton Based on studies carried out primarily in mammalian cells, the Rho-related GTPases have been grouped into three subfamilies, according to functional as- pects. Rae proteins are involved in the formation of lamellipodia and membrane ruffles, Rho members coordinate stress fibre and adhesion plaque forma- tion, and Cdc42 stimulates the formation of filopods (Hall 1994). Recent data indicate that besides regu- lation of the cytoskeleton organization, Rho-related GTPases are involved in a variety of other cell func- tions, including endocytosis, transcriptional regula- tion and growth control. Indeed, the pathways regu- lated by each of these GTPases are connected to each other as well as to the pathways of other GT- Pases, especially those involving Ras (Ridley 1996). Not all processes dependent on Rho-like proteins are present in Dictyostelium. These cells display an intense protrusive activity but do not have, for ex- ample, stress fibres and adhesion plaques, which largely simplifies studies on the role of Rho-like pro- teins. Nine Rho-related proteins have been identified so far 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 Family in the Dictyostelium Cytoskeleton Dictyostelium discoideum has emerged as a widely employed model to investigate basic questions of molecular and cell biology (Maeda et al. 1997). Three features make D. discoideum an attractive model organism in which to stUGy the components of the actin cytoskeleton, the relationships among them and the elements involved in their regulation. First, Dictyostelium exhibits a particular life cycle in which free living amebae aggregate in response to chemotactic signals to build up a multicellular fruit- ing body in a process that requires the integrity of the actin cytoskeleton. Second, despite their appar- ent simplicity, Dictyostelium amebae are equipped with a complex actin cytoskeleton that endows the cells with motile behavior comparable to that of leukocytes. And third, Dictyostelium is amenable to a variety of biochemical, and molecular and cell biol- ogy techniques that make multidisciplinary ap- proaches possible. In particular the ease of cultiva- tion facilitates the isolation of proteins associated with the actin network; furthermore, the genome can be easily manipulated by means of recombinant DNA techniques, the application which is vastly widening our understanding of the role of cytoskele- tal components. Much is known about actin itself and about actin associated proteins, their activities and subcellular localization, in particular in response to extra- and intracellular stimuli (Noegel and Luna 1995). From these studies it is clear that regulatory components are needed that allow a cell to decide where and when the cytoskeleton has to be rearranged in re- sponse to a particular stimulus. Although a com- plete picture on the connections between signal transduction pathways and reorganization of cyto- skeletal structures is missing, evidence is accumu- lating on the role of small GTP-binding proteins of the Ras superfamily in this process. Based on sequence homologies the Ras-related small 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 nuclear protein import, and Ras and Rho regulate signal transduction pathways linking plasma membrane receptors to either growth and differentiation re- sponses or to changes in the cytoskeletal organiza- tion respectively (Hall 1994). In this short review we will focus on the members of the Rho family of GT- Pases described so far in Dictyostelium and will refer to potential regulatory elements upstream as well as downstream of these proteins that have been recognized in the last few years (Table 1). Rho-Related GTPases and the Cytoskeleton Based on studies carried out primarily in mammalian cells, the Rho-related GTPases have been grouped into three subfamilies, according to functional as- pects. Rae proteins are involved in the formation of lamellipodia and membrane ruffles, Rho members coordinate stress fibre and adhesion plaque forma- tion, and Cdc42 stimulates the formation of filopods (Hall 1994). Recent data indicate that besides regu- lation of the cytoskeleton organization, Rho-related GTPases are involved in a variety of other cell func- tions, including endocytosis, transcriptional regula- tion and growth control. Indeed, the pathways regu- lated by each of these GTPases are connected to each other as well as to the pathways of other GT- Pases, especially those involving Ras (Ridley 1996). Not all processes dependent on Rho-like proteins are present in Dictyostelium. These cells display an intense protrusive activity but do not have, for ex- ample, stress fibres and adhesion plaques, which largely simplifies studies on the role of Rho-like pro- teins. Nine Rho-related proteins have been identified so far in Dictyostelium using a variety of approaches. Bush et al. (1993) used degenerated oligodeoxy-

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Page 1: 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-

Page 2: Small GTP-Binding Proteins of the Rho Family in the Dictyostelium Cytoskeleton

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

Page 3: Small GTP-Binding Proteins of the Rho Family in the Dictyostelium Cytoskeleton

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

Page 4: Small GTP-Binding Proteins of the Rho Family in the Dictyostelium Cytoskeleton

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-

Page 5: Small GTP-Binding Proteins of the Rho Family in the Dictyostelium Cytoskeleton

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]