Print version ISSN 0716-9760
Biol. Res. vol.35 no.2 Santiago 2002
Cytohesins and centaurins control subcellular trafficking
of macromolecular signaling complexes: Regulation by
phosphoinositides and ADP-Ribosylation Factors
MUHAMED A. HAWADLE, NAJEEM FOLARIN, ROSEMARY MARTIN AND TREVOR R. JACKSON*
Department of Haematology. Royal Free & University College Medical School. Rowland Hill Street. London, NW3 2PF, England
The ADP-ribosylation factor family of small GTP-binding proteins are implicated in the regulation of vesicular transport and control of cytoskeletal and cell adhesion events. The phosphoinositide 3-kinase, phosphoinositide 4-P 5-kinase and phospholipase D signaling pathways are major regulators of ARF signaling cascades. Two families of ARF regulatory molecules, the cytohesin ARF-Guanine nucleotide Exchange Factors and the centaurin GTPase-Activating Proteins provide key targets for the action of these lipid signals. A critical feature of the regulation of ARF signaling is coordinated recruitment of exchange factors, ARFs and GAPs to appropriate subcellular locations. These complexes drive repetitive cycles of ARF activation and membrane association that underlie the processes of cell movement as well as endosomal uptake and intracellular redistribution of signaling molecules. Cytohesins and centaurins bind specifically to a variety of other signaling proteins and these interactions may provide routes for regulated recruitment to the sites of ARF activation. Through their ability to control endosomal trafficking/recycling of these supramolecular signaling complexes ARF and phospholipid signaling pathways may have consequences that reach as far as the regulation of gene transcription and control of cell fate.
Key terms: ADP-ribosylation factor; Cytohesin; Centaurin; GTPase Activating Protein; Guanine nucleotide Exchange Factor; Phosphoinositide.
SMALL GTP-BINDING PROTEIN SIGNALIN
Small guanine nucleotide binding proteins, as typified by members of the Ras and Rho families are common components of the signaling machineries that regulate fundamental biological processes such as cell division, differentiation and survival. These proteins cycle between active, GTP-loaded and inactive-GDP loaded conformations in response to interactions with Guanine nucleotide Exchange Factors (GEFs) or GTPase Activating Proteins (GAPs). This regulated cycle allows the proteins to function as molecular switches whose activated/GTP-loaded forms interact with specific effector molecules to propagate information through a network of signaling pathways. The ADP-ribosylation factor proteins (ARFs) are a further family of Ras related proteins best characterized as regulators of membrane trafficking and organelle structure (Chavrier & Goud 1999). However, the ARFs are now increasingly gaining recognition as regulators of cytoskeletal structure and hence may function as components of signaling pathways regulating cell movement, morphology and differentiation (Jackson et al., 2000b). The means by which extracellular signals can regulate specific ARF signaling pathways and the part played by macromolecular complexes in regulating and propagating these events are the subject of our research and hence of this paper.
The ARF protein family
The ARFs were originally defined by their ability to act as co-factors for cholera toxin mediated ADP-ribosylation of the heterotrimeric GTP-binding protein Gs (Moss & Vaughan 1995). There are 6 mammalian isoforms divided into 3 subfamilies: ARF1, ARF2 and ARF3 belong to Class I, ARF4 and ARF5 make up Class II whilst ARF6 is the sole member of Class III. This classification reflects sequence homology and differences in apparent subcellular localisations (Chavrier & Goud 1999; Donaldson & Jackson 2000). Class I and II ARFs are generally associated with vesicle trafficking events in the Golgi while ARF6 is found mainly at the plasma membrane. ARFs are distinctive from other Ras family members in two ways: First, they have negligible basal GTPase activity making them entirely dependent on association with GAP proteins for the inactivation step of their cycling; and second, all possess an N-terminal myristoyl modification. In the inactive state, the myristoyl moiety nestles in a hydrophobic pocket on the protein surface but on GTP binding this pocket closes, excluding the myristoyl group (Pasqualato et al., 2001) leading to membrane association of activated ARF. This naturally suggests that the regulated activation and inactivation of ARF proteins must occur in the immediate vicinity of a membrane surface and that the effectors of ARF signaling are likely to also function in this environment (Figure 1).
|Fig. 1. A schematic representation of the process of membrane recruitment occurring during the activation/deactivation cycle of ARF-GTPases and the hypothesized role of these events in signal transduction.|
Biological processes regulated by ARF proteins:
ARF proteins have long been recognised as crucial regulators of vesicle trafficking events in the golgi-endoplasmic reticulum system, where GTP-bound ARF1 promotes coat protein assembly on donor membranes (Donaldson et al., 1992) and the subsequent budding off of coated vesicles. ARFs control membrane recruitment of many different vesicle coat proteins, including COPI, adaptor proteins AP-1, AP-3, AP-4 and the newly identified family of GGAs 1-3 (Golgi Associated, g-adaptin homologous, ARF-interacting proteins ) (Puertollano et al., 2001). The complexes formed between coat proteins and GTP-bound ARFs appear dynamic (Presley et al., 2002). Thus, recruitment and/or activation of an ARF-GAP may be a prerequisite for the release of ARF from the membrane and the subsequent uncoating events required for vesicle fusion with acceptor membranes (Teal et al., 1994; Tanigawa et al., 1993). However, recently attention has shifted away from the Golgi and towards other sites of ARF protein function. ARF1 promotes coat protein assembly on endosomal membranes (Moss and Vaughan 1995) and evidence has accumulated that ARF6 can influence both endosomal function and the assembly of actin-cytoskeletal complexes at the plasma membrane (Chavier and Goud, 1999; Turner et al., 2001; Jackson et al., 2000b). It is the recognition of these overlapping activities and their relationship to lipid signaling processes that has led to a new understanding of the place ARF-protein complexes occupy in the network of pathways that control the biology of the cell.
The cytohesin family of ARF-Guanine nucleotide Exchange Factors
The identification of ARNO, ARF Nucleotide binding site Opener, an ARF-GEF (Chardin et al., 1996) revealed it had a sequence almost identical to a previously identified integrin binding protein fragment, CTS1.8 and the extremely closely related cytohesin-1 (Kolanus et al., 1996). ARNO has also been named cytohesin 2 (Frank et al., 1998a) and has provided the first glimpse of a role for ARFs in cytoskeletal and adhesion signaling. A direct role in signaling was provided by the identification of a further cytohesin related protein GRP1 (General Receptor for Phosphoinositides1) capable of binding the lipid messenger PtdIns 3,4,5-P3 (Klarlund et al., 1997). These proteins share a core structure of a Sec7 homology domain encoding the ARF-GEF activity and c-terminal pleckstrin homology (PH) domain. This tandem arrangement of domains is present in a number of other ARF-GEF proteins but not in the much larger and evolutionarily more distant GEA1 and BFG ARF-GEFs (see Figure 2). We have suggested that on the basis of their evolutionary relationship the PH domain containing proteins can be treated as a superfamily termed the cytohesins though this view is not shared by all (see Derrien et al., 2002). There is considerable debate over whether the GEF activities of individual cytohesins shows specificity for ARF isoforms (reviewed in Jackson et al., 2000b) but in vitro and in vivo studies suggest that cytohesins 1-4 act on multiple ARFs. EFA6A/cytohesin5 and Tic/cytohesin6/EFA6B are reported to prefer ARF6 (Derrien et al., 2002; Franco et al., 1999). These differences may reflect the diversity in the spatial functional requirements of these proteins. Cytohesins 1-4 have been reported to be found in the cytoplasm, plasma membrane, golgi and on secretory granule membranes (see below) where multiple ARF species may be available, whereas cytohesin5/EFA6A and cytohesin6/Tic have so far been reported to associate with the plasma membrane and a recycling endosomal compartment in which ARF6 may be the only ARF substrate available (Derrien et al., 2002; Franco et al., 1999). However, our cell fractionation studies suggest that endogenously expressed Tic/cytohesin6 is present in a wider range of membrane compartments suggesting other locations might contain ARF6, that there remains a possibility of interaction with other ARFs, or that there may be ARF6 independent functions for this protein (See Figure 3).
Fig. 2. Schematic representation of the phylogeny and domain structures of ARF-Guanine nucleotide Exchange Factors. Gray branches indicate proposed subfamilies of ARF-GEFs, gray blocks represent modular domains within proteins. Those represented include ARF-Guanine Nucleotide Exchange Factor homology (ARF-GEF), pleckstrin homology (PH), and regions with a high probabability of forming coiled-coils (wavy shading). The scale bar represents amino acid substitution at a rate of 0.1 residue per position. An alignment of representatives of all eukaryotic ARF-GEF families was made using the DIALIGN 2 algorithm (http://bibiserv.techfak.uni-bielefeld.de/dialign/ and (Morgenstern 1999) and default parameters. The UPGAMA tree output was then visualized using TreeView (see http://taxonomy.zoology.gla.ac.uk/rod/treeview.html and (Page 1996). Conserved protein sequence motifs were detected by screening against the Protein families database of alignments and HMMs (http://www.sanger.ac.uk/Software/Pfam/index.shtml). Regions with a high probability for forming coiled-coils were predicted by use of the COILS algorithm (http://www.ch.embnet.org/software/COILS-form.html and (Lupas et al. 1991) with a window of 24. For the identities of the proteins aligned see Table I.
Fig. 3. ARF6 regulatory molecules Cytohesin6/Tic, Centaurin ß1 and ß2 are associated with plasma membrane, early endosomal and dense membranous fractions of U937 and HL60 cell lines. The figure shows the relative distribution of proteins in individual fractions as well as Post nuclear supernatant (PNS), soluble (S100) and total membrane (P100) fractions. Cell fractions were prepared by densitity gradient ultracentrifugation through an OptiprepTM gradient by a procedure modified from that desribed in (Caumont et al. 2000). Antisera used were against Cytohesin6/Tic:Tic70; centaurin ß1: J151/4; ß2: J149/3; for visualization of Integrin a5 and Early Endosomal Antigen 1 (EEA1) antibodies were purchased from Transduction Labs; antisera to Golgi associated protein p58 were from Sigma-Aldrich.
Phosphoinositide control of cytohesin ARF-GEFs: Regulation by location
The report that the PH domain of cytohesin-3/GRP1 could bind the lipid second messenger PtdInsP3 with high affinity and specificity was coupled with the realisation that this and other cytohesin PH domains shared a common consensus sequence capable of 3-phosphorylated inositol lipid binding (see Ferguson et al., 2000). Subsequently, PI 3-kinase-, and PH domain-dependent recruitment to the plasma membrane was reported for GRP1/cytohesin-3, cytohesin-1 and ARNO/cytohesin-2. However, confusingly others reported considerable levels of constitutively plasma membrane associated ARNO. These conflicting reports appear to have been resolved by two studies: One showed that alternative splicing can give rise to cytohesins 1-4 having either 2 or 3-glycine residues located in a loop between two beta-sheets which make up part of the inositol phosphate binding site in the PH domain of these proteins (Ogasawara et al., 2000; Cullen and Chardin, 2000). The other study revealed the significance of these `di-' and `tri-gly' isoforms. In cytohesin-2 and -3, the insertion of the extra residue markedly increases the affinity of the proteins for PtdIns 4,5-P2 but has little effect on their high affinity, 0.5-3 mM, binding of PtdInsP3. In the case of cytohesin-2, the relatively high affinity of the `tri-gly' PH-domain for PtdIns 4,5-P2, at about 3.7mM, is similar to that seen in the PH-domain of PLCd and sufficient to support constitutive association with the plasma membrane for a significant proportion of the cellular protein. The lower PtdIns 4,5-P2 binding affinity (120mM) of `di-gly' cytohesin-2 gives a cytoplasmic distribution unless sufficient PtdInsP3 accumulates to recruit the protein to sites of PI 3-kinase activation. The `di-' and `tri-gly' PH domains of cytohesin-3 show the same qualitative shift in specificity but their affinity for PtdIns 4,5-P2 remains too low to give constitutive membrane recruitment. The `tri-gly' cytohesin-1 PH domain shows around 3-fold higher preference for PtdInsP3 over PtdIns4,5-P2, but with lower overall affinity for either which may explain the inability of PI 3-K to mediate recruitment of isolated PH domain constructs (see Cullen & Chardin 2000; Klarlund et al., 2000). Full length `tri-gly'cytohesin-1 is recruited to PtdInsP3 containing membranes and it has been suggested that a polybasic region situated c-terminal to the PH domain in cytohesins 1-4 may assist membrane recruitment by providing additional interactions with acidic phospholipids (Santy et al., 1999; Nagel et al., 1998a). It may also provide a further site for regulation of these proteins (see below).
Binding of PtdInsP3 to cytohesin-3 has been proposed to result in allosteric activation of GEF activity but the effects reported are small and very dependent on the presence of detergents making their in vivo significance difficult to interpret (Klarlund et al., 1998).
The lipid binding characteristics of other cytohesins have not been reported but EFA6/cytohesin5 and Tic/Cytohesin6/EFA6B show significant levels of association with cell membranes. For Tic/Cytohesin6/EFA6B the PH domain is sufficient to direct association with plasma membrane microvilli and ruffles. These sites have been inferred to have high local PtdIns4,5-P2 levels, as determined by PLCd PH domain-chimera binding to these membrane regions. Deletion mutants of Tic/Cytohesin6/EFA6B lacking the PH domain show a cytoplasmic distribution and have no effect on cell morphology (Derrien et al., 2002) emphasizing that the function of the PH domain in these molecules is to provide appropriate membrane targeting via interaction with phosphoinositides and that this is crucial to their role in cell regulation.
Cyothesins in supramolecular complex assembly: I) Integrins and the cytoskeleton
Integrins are heterotrimeric cell adhesion molecules which function in bidirectional signaling carrying information on cell-cell/matrix contacts to the cell interior (Jackson et al., 2000a; Arthur et al., 2002; Avalos et al., 2002) and also transmitting signals for specific regulated adhesion events from the cytoplasm to the outside of the cell (so called outside-in and inside-out signaling pathways, Kolanus & Seed, 1997). The sequences of cytohesins-1 and -2 were identified by their ability to interact with the c-terminal domain of integrin ß2 indicating a possible role in the process of signaling complex assembly with the cytoplasmic tail of ß2 containing integrins (Kolanus et al., 1996). This interaction occurs between identified residues in the integrin tail and part of the highly conserved Sec7 domain of these cytohesins. The interaction between cytohesin-1 and the ß2 sequence results in an increase in the avidity of integrin-substrate binding, an event which normally accompanies stimulation of adherence by PI 3-kinase coupled receptors in ß2 integrin expressing white blood cells. The GEF activity of cytohesin-1 is not required for this effect indicating that protein-protein interaction alone can regulate this inside-out signaling event (Geiger et al., 2000). The presence of the PH domain is, however, crucial for this process and an isolated PH domain acts as a dominant negative inhibitor (Kolanus et al., 1996) demonstrating that correct localisation of the protein in response to the production of PtdInsP3 is critical for the protein-protein interaction to occur (Nagel et al., 1998b). A requirement for the cytohesin-1 GEF activity has emerged in the subsequent processes of cell spreading and movement (Geiger et al., 2000) and, consistent with the plasma membrane and peripheral endosomal localisation of the proposed ß2-integrin-cytohesin signaling complex, ARF6 appears to be the substrate of cytohesin-1 GEF activity (Weber et al., 2001). Cytohesin-3/GRP1 is also able to mediate regulation of cell attachment via ß2 integrins (Korthauer et al., 2000) and ARNO has been shown to regulate endothelial cell motility (Santy & Casanova 2001) suggesting that this may be a signaling function shared by each of the closely related cytohesins 1-4.
As indicated above, the polybasic regions of cytohesin-1 and 2 contribute to membrane recruitment but in cytohesins-1,2 and 4 this region also contains a potential site for phosphorylation by PKC. Phosphorylation by PKC is an event which has been demonstrated in vivo and in vitro for cytohesin-1 and -2 (Frank et al., 1998b; Dierks et al., 2001). It has been suggested that phosphorylation of this residue in cytohesin-2 provides an electrostatic switch to block membrane association providing an additional mechanism for regulation of interactions with cell membranes and other structures (Santy et al., 1999). However, phosphorylation of the equivalent site in cytohesin-1 does not alter in vitro lipid binding but, in vivo, leads to association with the cortical actin cytoskeleton and contributes to PMA stimulated cell adhesion (Dierks et al., 2001). Whether these findings represent differences important for the regulation and signaling of these cytohesins remains to be determined.
II) Coiled-coil interactions mediate membrane localisation of cytohesin binding partners
Outside of the conserved Sec7 and PH domains all cytohesins contain regions predicted to form coiled-coil (CC) structures often implicated in protein protein-interactions. Isolated CC regions of cytohesins 1-3 target eGFP expression to the Golgi (Lee & Pohajdak, 2000). This and a minority of other studies have reported constitutive Golgi association of cytohesins 1-3, as well as inhibition of secretion and Golgi disassembly on overexpression (Franco et al., 1998; Monier et al., 1998; Lee & Pohajdak 2000; Lee et al., 2000) leading to the suggestion that interactions between the CC domain and another factor might target these cytohesins to the Golgi. In fact, a number of distinct binding partners for this region of cytohesins 1-3 have now been identified (see Figure 4). The most recently identified is GRSP1 (GRP1 Signaling Partner 1) a FERM ( Band 4.1/Ezrin/Radixin/Moesin homology region; also found in FAK and JAK kinases) domain containing protein shown to bind to cytohesin-3 via an internal CC region. On co-expression in CHO cells, cytoplasmic GRSP1-cytohesin-3 complexes are recruited to plasma membrane ruffles following insulin stimulation (Klarlund et al., 2001). Intriguingly, GRSP1 is homologous to a genbank entry identified as Golgi associated band 4.1 like protein (Accession number BAB17031), leading to the speculation that under other conditions this interaction might explain the localisation reported above. The proteins GRASP (GRP1 Associated Protein)/Tamalin and Cybr/CASP (Cytohesin Binding Regulator/Cytohesin Associated Protein; also known as B3-1, Cytohesin- binding protein HE and PSCD-Binding Protein) contain PDZ domains, as well as CC regions, that mediate binding to their cytohesin partners (Kitano et al., 2002; Mansour et al., 2002; Nevrivy et al., 2000; Tang et al., 2002). GRASP is specific for cytohesins-2 and 3, whilst Cybr/CASP can bind to CC regions from Cytohesins 1-3. GRASP/Tamalin is a retinoic acid responsive gene product and overexpression leads to increased plasma membrane association of cytohesin-3 (Nevrivy et al., 2000). Cybr/CASP is a cytokine responsive gene originally identified in NK cells. Expressed singly in unstimulated Cos-1 cells Cybr/CASP localises to perinuclear tubulovesicular structures in close association with but distinct from the Golgi. Co-expression of Cybr/CASP with cytohesin-1 leads to redistribution from these structures into the cytoplasm and to colocalisation in membrane ruffles following EGF stimulation (Mansour et al., 2002). Cybr/CASP is also reported to enhance cytohesin-1 ARF-GEF activity in vitro (Tang et al., 2002). In the nervous system GRASP/Tamalin forms a complex between group 1 mGlu receptors and cytohesin-2 which is enriched in postsynaptic membrane fractions where it may participate in distribution of these receptors to neurites (Kitano et al., 2002). The phorbol ester target Munc13A is also a binding partner of Cytohesin-1 (see also (Jackson et al., 2000a; Wright & McMaster 2002). Both proteins are associated with regions of high membrane turnover in the presynaptic transmitter release zones (Zaal et al., 1999). Roles for cytohesins-1 and-2 in mediating increases in plasma membrane associated ARF6 have previously been reported in distinct neurosecretory events (Ashery et al., 1999; Caumont et al., 2000) but, whether this involves formation of a larger complex, though likely, remains undetermined.
|Fig. 4. Schematic representation of protein binding partners identified for small Cytohesins (1-4). Known cytohesin interacting proteins are represented with domain structures. Known sites of interaction are indicated by solid double-headed arrows. A potential Coiled-coil site of interaction in Munc13A is indicated with a dotted arrow. See text for a full description of these interactions. Protein domains identified in cytohesin binding proteins include PDZ, PKC conserved domains C1 and C2, and an Alanine/Proline rich-region (AR) in Tamalin/GRASP.|
No binding partners have been identified for other cytohesins but their sequences contain CC and sometimes also proline-rich regions that may form binding sites for other CC or SH3 domain containing proteins. The observation that PH domain mediated membrane targeting of the CC region of Tic/cytohesin6/EFA6B is sufficient to drive microvillar elongation suggests that this region may provide a binding site for a cytoskeletal regulator (Derrien et al., 2002).
Cycles of ARF activation and the need for ARF-GTPase Activating Proteins:
ARF proteins are wholly dependent on extrinsic GTPase Activating Proteins to drive GTP hydrolysis. This event is necessary for release from membrane association and/or effector complexes. Furthermore, there is evidence to suggest that in systems including cell spreading/movement and vesicle trafficking, there is an ongoing cycle of arf activation-deactivation requiring the presence of both GEF and GAP activities in the same membrane compartment. The purification and cloning of ARF-GAP1 gave the first picture of the structure of one of these proteins. The GAP function of this protein is located in a region approximately 120 amino acids in length with a characteristic Zn2+ binding finger located at the n-terminal of the domain (Goldberg, 1999). Our efforts and those of a number of co-workers using biochemistry and genomics have identified a `superfamily' of confirmed and putative ARF-GAPs whose structures and proposed evolutionary relationships are shown in Figure 5. Unfortunately, the competitive nature of this field means that a single protein or its isologs can have many different names and so the literature describing them is complex and confusing. At the time of writing, the ARF-GAP community has begun a debate that may resolve this situation by adopting a common nomenclature. However, in the absence of this consensus, we will continue to use the system detailed in Tables II (A-E).
Fig. 5. Schematic representation of the phylogeny and domain structures of ARF-GTPase Activating Proteins. Gray branches indicate proposed subfamilies of ARF-GAPs, gray blocks represent modular domains within proteins. Those represented include ARF-GTPase Activating Protein (ARF-GAP), Rho- GTPase Activating Protein (Rho-GAP), pleckstrin homology (PH), Ankyrin repeats (A), Src Homologous region 3 (SH3), Ras binding site (R), Sterile Alpha Motif (SAM), Rab GTPase-like (Rab), GIT (G-protein Interacting ARF-GAP) homology and BAR (Amphyphysin and clathrin binding protein homologous) domains as well as regions with a high probabability of forming coiled-coils (wavy shading). The scale bar represents amino acid substitution at a rate of 0.1 residue per position. Alignment and analysis was performed as described in the legend to Figure 2. For the identities of proteins aligned see Tables II (A-E).
Regulation of ARF-GAP function: Lipid binding can control membrane location and GAP activity
Given that their substrates, GTP-loaded ARF proteins, are likely to be found in membrane associated complexes it is little surprise that potential lipid binding motifs occur in many members of the ARF-GAP superfamily. The most common potential lipid-binding domain found in association with ARF-GAP homologous sequences is the PH domain (Figure 5). We identified the ARF-GAP homology and PH domain containing protein, centaurin a1, as a high affinity PtdInsP3 binding protein (Hammonds-Odie et al., 1996). We have proposed that ARF-GAP sequences that contain PH domains can be also termed centaurins and some aspects of their role in cell regulation are presented here though a more complete review can be found in (Jackson et al., 2000b). Other lipid binding ARF-GAPs include the PtdInsP3 regulated GIT1 (Vitale et al., 2000) and ZAC, from Arabidopsis, which shows membrane association in vivo and contains a non-specific phospholipid binding C2 domain and an unidentified PtdIns 3-P binding site (Jensen et al., 2000). ARF-GAP1 shows localisation to the Golgi (Cukierman et al., 1995) where it may be regulated by interaction with phosphoinositides and diacylglycerols as well as protein-protein interactions (Antonny et al., 1997; Szafer et al., 2000; 2001).
Phosphoinositide regulation of centaurin ARF-GAPs
Centaurin a isoforms have an N-terminal ARF-GAP domain followed by paired PH domains. In a1 the sequence of each PH domain contains potential 3-phosphoinositide binding sequences and the protein binds to PtdInsP3 with high affinity (Ferguson et al., 2000; Hammonds-Odie et al., 1996; Venkateswarlu et al., 1999). Exogenous tagged-centaurin a1 shows PI 3-kinase mediated recruitment to plasma membrane ruffles, mediated predominantly by the more c-terminal PH domain (Venkateswarlu et al., 1999). Live imaging reveals that eGFP-centaurin a1 also associates with internalising `endosomal' globular and tubular structures (Figure 6). In most cells investigated, exogenous and endogenous centaurin a1 is also found in the nucleus (see Figure 6 and Venkateswarlu et al., 1999), where one report also demonstrates recruitment to PtdInsP3 following activation of nuclear membrane associated PI 3-kinase (Tanaka et al., 1997). The PI 3-kinase dependent recruitment of centaurin a1 to plasma membrane ruffles and endosomes indicates that it will co-localise with cytohesins and ARFs. Surprisingly though, centaurin a1 does not show GAP activity against ARF proteins or close relatives leading to the suggestion that it may provide an ARF effector function. Centaurin a2 shows constitutive membrane association, but whether this results from the lack of discrimination between PtdInsP3 and PtdIns 4,5-P2 exhibited by the PH domains (Whitley et al., 2002) or the presence of a putative N-terminal myristoylation site remains undetermined.
|Fig. 6. Exogenously expressed eGFP-Centaurin a1 is recruited to plasma membrane ruffles as well as endosome and tubulovesicular structures in response to Insulin stimulation. PC3 cells grown on glass coverslips were transiently transfected with pEGFPC1-RnCentaurin a1 and subject to serum starvation prior to imaging. The 60 second frame shows a pair of unstimulated cells expressing low and moderate levels of EGFP-RnCentaurin a1, respectively. Insulin was added at approximately 90 seconds and subsequent cell responses include the formation of plasma membrane ruffles highly enriched in the centaurin a1 chimera (white arrowheads). In the upper cell the fluorescent protein can also be seen decorating a tubulovesicular structure (t) that extends between the cell body and the region of ruffling in the upper extremity of the cell. This structure was visible for around 3 minutes before appearing to fragment into a number of vesicular structures (430s) that may be endosomal in nature (e). Images were obtained using a PerkinElmer Ultraview confocal system mounted on an Olympus IX70 microscope using a 40x oil immersion objective. The Kr/Ar laser was maintained on minimum power and cells were held at 37oC using a Perkin Elmer environmental control system.|
The mammalian ß-centaurins are a subfamily sharing a common core structure made up of a single PH domain followed by ARF-GAP and Ankyrin repeat regions. All have N-termini containing one or more CC regions potentially involved in protein-protein interactions, centaurins ß3 and ß4 also have C-terminal SH3 and proline rich domains responsible for interaction with other proteins. We have shown that in vitro centaurin ß4 can bind to products of PI 3-kinase or PtdIns 4,5-P2 (Kam et al., 2000) and recruitment to these lipids may in part be responsible for plasma membrane localisation seen on cell stimulation (Brown et al., 1998; Jackson et al., 2000a; Randazzo et al., 2000). Centaurins ß1 and ß2 are recruited to plasma membrane ruffles in response to cell stimulation by growth factors and insulin where they colocalise with ARF6 and cytohesins (T.R.Jackson, in preparation; and Jackson et al., 2000a). All ß-centaurins tested are dependent on phospholipids in order to show ARF-GAP activity. For centaurins ß1,2 and 4 this includes a requirement for phosphatidic acid (PA) as well as polyphosphoinositides (Andreev et al., 1999; Jackson et al., 2000a; Kam et al., 2000). Allosteric activation of the ß4 ARF-GAP shows a preference for PtdIns 4,5-P2 over 3-phosphorylated lipids though both appear to bind to overlapping sites in the PH domain (Kam et al., 2000). The requirement for PtdIns 4,5-P2 and PA for ß-centaurins to show ARF-GAP activity suggests that they can be co-ordinated by the products of GTP-ARF regulated enzymes, PtdIns 4-P 5-kinase and PLD. PLD is also a target of negative regulation by a-actinin and gelsolin leading to the possibility that centaurins are conditional ARF-GAPs sensitive to both positive and negative feedback from ARF and cytoskeletal signaling pathways (Turner et al., 2001).
Centaurins ß1 and ß2 show a preference for ARF6, whilst ß3 and ß4 instead have higher activity on class I and II proteins (Andreev et al., 1999; Brown et al., 1998; Jackson et al., 2000a; Kondo et al., 2000). These in vitro substrate specificities correlate with in vivo ability of centaurins ß1 and ß2, but not ß4, to block ARF6 specific cytoskeletal responses. However, all 3 of these ß centaurins can block PDGF stimulated dorsal ruffling, suggesting that distinct cytoskeletal response may require inputs from differing and/or multiple ARF signaling pathways (Jackson et al., 2000a).
The PH domains of the g centaurins lack the phosphoinositide-binding consensus seen in other family members and our imaging of eGFP-centaurin g1 (T.R. Jackson, unpublished results) indicates that it remains cytoplasmic during insulin stimulated ruffling responses. However, an alternative protein product of the centaurin g1 locus is reported to participate in growth factor regulation of nuclear PI 3-kinase activity (Ye et al., 2000). The d centaurins have multiple PH domains, demonstrate PI 3-kinase mediated membrane recruitment, and show PtdInsP3/PtdIns3,4-P2 regulated ARF-GAP activities. In addition, these proteins also have Rho-GAP activity. Their potential function in a multimolecular complex integrating/regulating these two signaling pathways is reviewed elsewhere (Santy & Casanova 2002).
The centaurins as components of macromolecular complexes
SH3 domain containing proteins bind to class I and II SH3 target sequences in ß3 and ß4 centaurins. Partners found in vitro include Crk and Lck SH3 domains (Brown et al., 1998; King et al., 1999); the Src SH3 domain also binds in vitro and activated Src binds and phosphorylates both centaurins ß3 and ß4 (Brown et al., 1998; Kondo et al., 2000) though the biological significance of this event is unclear. The SH3 domains present in centaurins ß3 and ß4 bind Pyk2 and FAK tyrosine kinases, respectively (Andreev et al., 1999; Liu et al., 2002) and this region also directly associates with paxillin (Kondo et al., 2000). All of these proteins are implicated in macromolecular complexes associated with integrin containing focal adhesions (Giancotti, 2000; see also Jackson et al., 2000a; Arthur et al., 2002; Avalos et al., 2002) leading to the suggestion that these centaurins might cooperate with cytohesins to regulate cytoskeletal reorganisation during cell movement.
Overexpression of centaurin ß3 or ß4 reduces cell spreading and inhibits the appearance of paxillin and FAK rich focal adhesions (Furman et al., 2002; Liu et al., 2002; Randazzo et al., 2000); an effect which is diminished in mutants lacking the C-terminal SH3 domain (Liu et al., 2002) suggesting that SH3 domain containing ß-centaurin proteins may regulate the subcellular distribution of Focal Adhesion Kinases. Growth factor stimulation recruits centaurins ß1-4 to the plasma membrane, but from differing starting locations. In resting cells, ß1 and ß2 are cytoplasmic (Figure 7 and Jackson et al., 2000a), while ß3 redistributes from the perinuclear region and ß4 from peripheral focal adhesions. In the latter two cases, these resting distributions resemble those of the Focal Adhesion Kinases they associate with (Andreev et al., 1999; Liu et al., 2002; Randazzo et al., 2000). The redistribution of centaurin ß3, on stimulation, also corresponds to the path followed by paxillin and associated proteins during the assembly of nascent focal adhesions, a process that is dependent on activation of ARF1 in the Golgi (Turner et al., 2001b).
Fig. 7. A schematic model to illustrate the possible roles of ARF6/centaurinß1 and ARF1/centaurin ß3 pathways in the delivery and retrieval of membrane, cytoskeletal and cell adhesion complex regulatory molecules during lamellipodial extension. For a full explanation see text. FA- focal adhesion; EE - early endosome; RE - recycling endosome.
The persistence of peripheral plasma membrane ruffle-associated ß3- and ß4-proteins on overexpression is correlated with decreased mature focal adhesion formation and consequent cell spreading, suggesting that increased ARF nucleotide cycling opposes stable adhesion/spreading.
This finding is supported by two studies suggesting that dominant negative GAP mutants lacking GAP activity show reduced inhibitory effects (Randazzo et al., 2000; Liu et al., 2002). Overexpression of centaurin ß3 is reported to increase chemotactic and chemokinetic responses to growth factors leading to the suggestion that regulation of the level/distribution of ß-centaurins may be associated with increased cell motility (Furman et al., 2002). However, these results contradict those found in monocytes (Kondo et al., 2000) suggesting cell type and/or the presence of other signaling components may alter the effects of ß-centaurins on cell movement and morphology.
All ß, g and d centaurins contain additional potential sites of protein-protein interaction, including proline rich repeats, CC, SAM (Sterile Alpha Motif) and Rab (Ras binding) domains (Figure 5). The determination of their partners will surely add further to our understanding of cell regulation.
Other targets of ARF and centaurin protein signaling: The endosome and beyond?
Endogenous or exogenously expressed centaurins ß1 and ß2 can associate with peripheral tubular endosomal structures; the appearance of these structures is enhanced by AlF4 (Aluminum Fluoride) treatment, but blocked by expression of activated ARF6, which appears to `trap' the proteins at the plasma membrane. Dominant negative ARF6 blocks association of ß1 or ß2 with either plasma membranes or endosomes suggesting that ARF-cycling is required for this accumulation to occur (Jackson et al., 2000a). We have also demonstrated the presence of centaurins ß1 and ß2 in cellular fractions including early endosomal and denser membranous compartments (Figure 3). The structures we have observed and this subcellular localisation resembles a tubulovesicular, RME-1/EHD1 (Eps15 Homology Domain1) containing, compartment implicated in plasma membrane recycling of proteins endocytosed by separate clathrin-dependent and indepent routes (Caplan et al., 2002). This compartment may correspond with, or alternatively be a derivative of the previously characterised Recycling Endosome (Radhakrishna & Donaldson 1997). ARF proteins are also regulators of lipid signaling enzymes of the Phospholipase D and Phospatidylinositol 4-P 5-kinase families and there is evidence that PtdIns 4,5P2 production may also be necessary for the cycling of ARF6 which supports these tubular endosomal structures (Brown et al., 2001). These structures are disrupted by overexpression of centaurin ß1 supporting a role for this ARF-GAP and appropriate phospholipid regulators in the control of ARF6-GTPase cycling required for formation of this compartment (Caplan et al., 2002).
Observations concerning centaurin a1 in `endosomal' compartments of PC12 cells and evidence of association between centaurin a1 and ARF6 (A.B.Theibert and T.R. Jackson in preparation), have led us to suggest that like ß centaurins, the a1 protein may associate with an endosomal compartment. Live cell imaging of Insulin stimulated PC3 cells expressing exogenous eGFP-Centaurin a1 shows that it rapidly associates with plasma membrane ruffles (arrowheads) as well as with vesicular endosomal (e) and tubulovesicular (t) compartments (Figure 6). In the absence of GAP activity, it is possible that centaurin a1 is participating as an effector, either of endosome formation and movement, or to direct subsequent sorting of signaling complexes to other cellular destinations. Centaurin a1 is also a binding partner for CK1 isoforms, a family of protein kinases implicated, amongst other things, in endocytic trafficking in mammals and yeast (Dubois et al., 2001; 2002). CK1 isoforms are also implicated in regulation of nuclear partitioning and transcriptional events and it is tempting to speculate that centaurin a1 might direct nuclear trafficking of CK1 via a sorting/signaling endosome to the nucleus. Pyk2 kinase has also recently been reported to be present in the nucleus where we have also detected centaurin ß3 and ß4 by imaging and/or western blotting. Centaurin ß3 is implicated in insulin signaling processes leading to adipocyte differentiation, a function which may reflect a wider role for centaurins in pathways controlling gene expression and cell fate (King et al., 1999).
A Nuffield Foundation Summer Research Bursary supported RM. The Children's Leukaemia Trust has supported MH and NF. A Children's Leukaemia Trust Senior Lectureship supported TRJ. Imaging was performed on a PerkinElmer Ultraview Confocal Microscope purchased with funds from the MRC, Children's Leukaemia Trust and the University of London Central Research Fund. Michael Nicklin kindly provided antisera to Tic/cytohesin6.
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Corresponding author: Trevor R Jackson. Department of Haematology, Royal Free &University College Medical School. Rowland Hill Street. London, NW3 2PF, England. tel: +44 20 7472 6315. lab: +44 20 7794 0500 x 3324. fax: +44 20 7830 2092
*Present address: (From 01 October 2002): Departments of Physiology and Dermatology. Floor 2 Leech Building. The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, England
Received: July 05, 2002. In revised form: July 10, 2002. Accepted: July 19, 2002