Rho/RacGAPs

Abstract

Regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) determine the activity of small GTPases. In the Rho/Rac family, the number of GEFs and GAPs largely exceeds the number of small GTPases, raising the question of specific or overlapping functions. In our recent study we investigated the first time ARHGAP25 at the protein level, determined its activity as RacGAP and showed its involvement in phagocytosis. With the discovery of ARHGAP25, the number of RacGAPs described in phagocytes is increased to six. We provide data that indicate the specific functions of selected Rho/RacGAPs and we show an example of differential regulation of a Rho/Rac family GAP by different kinases. We propose that the abundance of Rho/Rac family GAPs is an important element of the fine spatiotemporal regulation of diverse cellular functions.

Introduction

Members of the Rho family of small GTPases—Rho, Rac and Cdc42 being the most prominent—have a central role in many cell activities such as shape changes, motility, cytokinesis, muscle contraction or production of toxic oxygen metabolites. In their active, GTP-bound form Rho/Rac proteins initiate the formation of large molecular complexes and/or activate protein or lipid modifying enzymes. The proportion of GTP- to GDP-bound form of small GTPases is primarily dependent on the ratio of the activity of two types of regulatory proteins: guanine nucleotide exchange factors (GEFs) that favor binding of GTP to the protein and GTPase activating proteins (GAPs) that accelerate the endogenous GTPase activity and promote the GDP-bound, inactive form. In case of the Rho family, the guanine nucleotide dissociation inhibitors (GDIs) represent an additional group of regulatory proteins that sequester the GTPases in an inactive form in the cytosol. In addition, prenyl transferases play a significant role in determining the localization of small GTPases as well as their interactions with various regulatory proteins. Thus, there are multiple mechanisms that can lead to local increase of the active form of Rho/Rac proteins and this can also happen without any change in the activity of a GEF.

In case of the Rho family, GEFs and GAPs largely outnumber the small GTPases. Based on sequence homology, the number of potential Rho/Rac family GAPs was estimated to be around 70.^1-3 However, many of these proteins have never been expressed, and a few expressed proteins turned out not to have any GAP activity.^4,5 Thus, the real number of functioning Rho family GAPs is presently unknown. The abundance of GAPs acting on Rho family proteins raises the serious question about specific or overlapping functions. Our recent results contribute new information on this matter.

ARHGAP25: A Newly Identified RacGAP

We have expressed the first time ARHGAP25 protein and produced polyclonal antibodies against it.⁶ Immunoblotting supported the earlier in silico data about the tissue distribution of ARHGAP25:⁷ it is highly expressed in all types of human leucocytes. In neutrophilic granulocytes it is present both in the cytosolic and in the membrane fractions.

We investigated the GAP activity of ARHGAP25 and showed that from the major Rho-family members it reacts only with Rac but neither with Rho or Cdc42. In this property it is similar to BCR,⁸ but different from p50RhoGAP and p190GAP, that are the Rho-family GAPs demonstrated earlier to be present in human neutrophils.⁹

The domain structure predicted from the amino acid sequence of ARHGAP25 (Fig. 1) consists of an N-terminal PH-domain followed by the GAP domain, an interdomain stretch and a C-terminal coiled coil domain. We prepared several fragments of the protein and confirmed the localization of the GAP domain. We also determined Arg192 to be critical for the GAP-activity. We did not observe any difference between the RacGAP activity of the full-length protein and various fragments containing the GAP-domain. This observation is at variance with our earlier findings on p50RhoGAP,¹⁰ and indicate that ARHGAP25 probably is not in an autoinhibited state.

Figure 1. Domain structure of selected GAPs with verified RacGAP activity. In case of p190-A the phosphorylation sites, discussed in this paper, are also indicated. Abbreviations: PH, pleckstrin homology domain; CC, coiled coil; SH3, Src Homology 3 domain; C2, calcium-dependent lipid binding domain; P, proline-rich domain; S/T kinase, serine/threonine kinase domain; Sec14, Sec14-like domain; SH2, Src Homology 2 domain; C1, cysteine-rich phorbol ester Binding domain; FF, domains with two conserved phenylalanine residues; PBR, polybasic region; (According to the review of Tcherkezian et al.³)

The domain structure of ARHGAP25 resembles that of FilGAP (Filamin-A-associated RhoGAP, named also Rac1- and Cdc42-specific GTPase activating protein of 72 kDa, p73RhoGAP, or ARHGAP24) that was shown to bind specifically to the cytoskeletal protein filamin A and participate in regulation of cell polarity. FilGAP reacts both with Rac and Cdc42.¹¹ The sequence homology between ARHGAP25 and FilGAP is approximately 50%.

We have downregulated the level of ARHGAP25 in promyelocytic PLB cells and macrophages differentiated from primary monocytes, and expressed it in COSPhoxFcγR cells and obtained opposing effect on phagocytosis. Expression of ARHGAP25 inhibited, whereas silencing of the protein increased phagocytosis of opsonized particles. Thus, ARHGAP25 is a significant regulator of the phagocytic process.

With the discovery of ARHGAP25, the number of GAPs acting on Rac in human neutrophils is increased to 4 (BCR, p50GAP and p190GAP), and two more proteins (ABR and ARHGAP15) have been indicated to play a role in mouse neutrophils.^8,12 Thus, the general question on specific or overlapping functions is relevant also in neutrophils.

Specific or Overlapping Function of Rho/RacGAPs?

Although it is too early to give a definitive answer to this question, available data allow some speculations. Rho/RacGAPs are complex, multidomain proteins (Fig. 1) suggesting distinct molecular interactions and regulation. Indeed, there are data on specific interaction of a Rho/RacGAP with a plasma membrane receptor such as the binding of α2-chimaerin to the guidance receptor EphA4^13,14 or association of p190RhoGAP with plexin A1 and B1,¹⁵ or oligophrenin1 with the AMPA receptor subunits GluR1 and GluR2.¹⁶ Some GAPs were shown to interact specifically with cytoskeletal or junctional proteins such as FilGAP with filamin A¹¹ and p190GAP with p120catenin.¹⁷ It is thus probable that—similar to many GEFs—GAPs also participate in large molecular complexes where replacement of one GAP molecule with another GAP of different structure would not be feasible. This view is supported by the remarkable phenotypes that arise in the case of defective function of one specific GAP whereas several other GAPs of the same substrate specificity are expressed in the given cell (for details see Ligeti et al.¹⁸).

Some of our recent experiments also indicate specific involvement of RacGAPs in regulation of neutrophil functions.⁶ COSPhoxFcγR cells provide an excellent model for neutrophilic granulocytes: they possess all the subunits of the NADPH oxidase and FcγRIIa, and phagocytose Ig-coated particles.^19,20 COSPhoxFcγR cells do not express either ARHGAP25 or p50RhoGAP. We expressed these two proteins to similar levels in COSPhoxFcγR cells. Both proteins have clear RacGAP activity under in vitro conditions, and both proteins prevented the EGF-induced ruffling, a phenomen dependent on intracellular Rac activation.²¹ Nevertheless, only expression of ARHGAP25 interfered with phagocytosis, whereas the presence of p50RhoGAP did not influence the engulfment of opsonized yeast particles. Phagocytosis is a complex process and the molecular interacting partners of ARHGAP25 have yet to be identified.

Another Rac-dependent process in neutrophils is superoxide production. The active NADPH oxidase is assembled from 5 subunits. One of the essential subunits is Rac and for sustained transmembrane electron flow, Rac has to be present in the active, GTP-bound form.²² Addition of several GAPs with RacGAP activity (BCR, p50RhoGAP or p190RhoGAP) resulted in a decrease of the rate of superoxide production in a cell-free activation system.^9,23 Observations on RacGAP-deficient mice indicated that by constitutive downregulation of Rac activity, RacGAP(s) exert constant control over superoxide production. Neutrophils isolated from RacGAP-deficient animals produced more superoxide than control cells, resulting in severe intestinal necrosis following endotoxin stimulation.²⁴ Depletion of BCR or ABR, two RacGAPs with high homology, resulted in similar phenotypes that were more accentuated in double-knockout animals, suggesting that the two proteins could have—at least partially—overlapping functions.^8,25 Ongoing experiments in our laboratory with specifically GAP-depleted neutrophil membranes also indicate that the control of the NADPH oxidase may be shared between several RacGAPs (Lőrincz and Ligeti, unpublished observations).

Complex Regulation of GAPs

The varied domain structure of Rho/RacGAPs offers the possibility of distinct regulation. In addition to chemical mechanisms such as phosphorylation by several kinases or dephosphorylation by phosphatases, prenylation, ubiquitination, protein-protein or protein lipid interactions, also physical factors such as membrane curvature or mechanical stress have been shown to alter the activity of some GAPs.^26-28 Among the Rho/RacGAPs, p190RhoGAP (referred to as p190-A) is the most investigated protein, thus we use this example to enlighten the complexity of regulatory processes. P190-A has been shown to be phosphorylated at several sites by different kinases and to interact with different proteins (Fig. 1).

Starting from the C-terminal group, S1472, S1476, T1480 and S1483 are involved in the phosphorylation by glycogen synthase-kinase-3-β (GSK3β), that requires priming by ERK or p38 MAPK.²⁹ Both the priming and the main phosphorylation decrease the activity of p190-A toward both of its substrates, Rac and Rho (Fig. 2A,B). Experiments performed on fibroblasts or COS cells have shown cellular relevance of both phosphorylation steps.^29,30

Figure 2. Differential effect of phosphorylation of p190-A by GSK3β (A and B) or PKCα (C and D) on the substrates Rho and Rac. (A and B). Both priming with cytosol and phosphorylation by GSK3β result in a decrease of the RhoGAP and RacGAP activity of p190-A. (C and D) Phosphorylation by PKCα reverses the modifying effect of acidic phospholipids and renders p190-A a better RhoGAP but a weaker RacGAP.

Phosphorylation by PKCα occurs on 3 sites, S1221, T1226, and S1236, within a polybasic region on the N-terminal side of the GAP domain (Fig. 1). This phosphorylation per se does not modify the in vitro GAP activity of p190-A, but phosphorylation on S1221 or T1226 changes the localization of the protein due to a decrease of the electrostatic interaction of the polybasic domain (PBR) with acidic phospholipids.³¹ By doing this, PKC-phosphorylation reverses the substrate specificity modifying effect of acidic phospholipids.³² As a result, the preference of p190-A alternates between Rho and Rac, depending on the local conditions: in non-phosphorylated state the polybasic domain is attached to acidic phospholipids and the protein behaves more as a RacGAP, whereas upon PKC-phosphorylation, the polybasic domain is detached from the membrane and the protein acts more as a RhoGAP (Fig. 2C,D). Transfection of COS-7 cells with full-length or PBR-deficient or phosphorylation-deficient p190-A resulted in largely different morphology, indicating the cellular relevance of PKC-phosphorylation driven alteration of the substrate preference.³¹ Ongoing experiments support the physiological significance of the polybasic region in directing Rac- or Rho-dependent cellular processes (Lévay and Ligeti unpublished observations).

Tyrosine 1105 was the first amino acid identified in p190-A as a target of Src and Abl kinases activated via EGFR or integrin signaling.^33,34 This phosphorylation initiates the binding of p120RasGAP to p190-A and promotes their translocation to the plasma membrane.^35-38 Under in vitro conditions, neither phosphorylation of Y1105 nor binding of p120RasGAP had any influence on the GAP activity of p190-A.^39,40 In contrast, in cells stimulated by EGF or integrins or overexpressing Src kinases, T1105 phosphorylation of p190-A correlated with a decrease of Rho activity, translocation of p190-A to the plasma membrane and local disassembly of actin stress fibers, a reaction typically dependent on Rho activity.^34,37-41 Tyrosine phosphorylation of p190-A also resulted in neurite outgrowth, another indicator of low Rho activity.⁴¹ It was suggested that Tyr-phosphorylation followed by association with p120RasGAP allowed recruitment of p190-A to an environment favoring its interaction with its substrate.⁴⁰

In contrast to phosphorylation of Tyr1105, which initiated association of p190-A with p120RasGAP, growth-factor induced phosphorylation of p190-A on Tyr308 was shown to disrupt the interaction of the serum-responsive transcriptional regulator TFII-I with one of the FF-domains of p190-A. TFII-I released from phosphorylated p190-A translocated to the nucleus where it enhanced c-fos promoter activity.⁴² The effect— if any—of this protein-protein interaction upon GAP activity of p190-A has not been explored.

Taken together, the revealed details of regulation of p190-A indicate the multitude of possibilities: phosphorylation of the GAP may result in direct change of activity or indirect effect due to change of localization and environment, or no alteration of the GAP activity. Thus, the mere fact of phosphorylation does not allow any conclusion on the functional consequences.

Conclusion

Our recently published findings have added one more protein to the long list of characterized Rho/RacGAPs and accentuate the question: why has Nature devised so many of these proteins? Are they really necessary or is it a shear luxury? We believe that many of the Rho/RacGAPs are embedded in well-defined molecular complexes rendering them irreplaceable. In other cases, several GAPs seem to be involved in the regulation of the same biological function. This may just reflect the lack of our knowledge on the details of their molecular interactions, or may represent a true overlap in their functions. However, even in those cases where more than one GAP should be involved in the control of the same reaction, their regulation is expected to be rather different allowing selective modulation via different signaling pathways. The plethora of Rho/RacGAPs can thus be regarded as the molecular basis of fine spatiotemporal regulation of a wide variety of cellular functions.

Csépányi-Kömi R, Sirokmány G, Geiszt M, Ligeti E. ARHGAP25, a novel Rac GTPase-activating protein, regulates phagocytosis in human neutrophilic granulocytes. Blood. 2012;119:573–82. doi: 10.1182/blood-2010-12-324053.