Hematopoietic-specific Rho GTPases Rac2 and RhoH and human blood disorders

Abstract

The small guanosine triphosphotases (GTPases) Rho proteins are members of the Ras-like superfamily. Similar to Ras, most Rho GTPases cycle between active GTP-bound, and inactive GDP-bound conformations and act as molecular switches that control multiple cellular functions. While most Rho GTPases are expressed widely, the expression of Rac2 and RhoH are restricted to hematopoietic cells. RhoH is an atypical GTPase that lacks GTPase activity and remains in the active conformation. The generation of mouse knock-out lines has led to new understanding of the functions of both of these proteins in blood cells. The phenotype of these mice also led to the identification of mutations in human RAC2 and RHOH genes and the role of these proteins in immunodeficiency diseases. This review outlines the basic biology of Rho GTPases, focusing on Rac and RhoH and summarizes human diseases associated with mutations of these genes.

Keywords: Cytoskeleton; Hematopoiesis; Rac; Rho GTPases; RhoH.

Figure 1. Involvement of Rac in phagocytic cell function and RhoH in T cell receptor signaling

Figure 1A. Involvement of Rac in NADPH oxidase activation and superoxide production and cell migration. Inactive GDP and RhoGDP inhibitor(RhoGDI)-bound Rac is located in the cytoplasm. Activation signals mediated by formylmethionyl-leucyl-phenylalanine (fMLP) binding to its G-coupled receptor, results in dissociation of RhoGDI from Rac and subsequent cycling of Rac to the GTP-bound, active form. Together with p40^phox, p47^phox and the p67^phox subunit, activated Rac translocates to the cell or phagosomal membrane to form the active NADPH oxidase complex together with the membrane-bound proteins large glycosylated protein (gp91^phox) and the smaller adaptor protein p22^phox. Subsequent electron transfer from NADPH to molecular oxygen via flavin adenine dinucleotide (FAD) leads to formation of superoxide (O2-) radicals. It is postulated that phosphorylation of p47^phox and p67^phox is mediated by protein kinase C (PKC) and that PKC is also involved in phosphorylation and dissociation of RhoGDI from Rac and thus regulates Rac activation. Activated, GTP-bound Rac also regulates cytoskeleton reorganization via downstream mediators of interaction with F-actin. Lack of Rac therefore disrupts formation of the phagocytic oxidase complex and inhibits migration in phagocytes. (Figure adapted from). Figure 1B. Involvement of RhoH in T cell receptor signaling. The T cell receptor (TCR) complex consists of TCR chains, CD3 and ξ-chain accessory molecules and the co-receptor CD4 or CD8. RhoH is required for phosphorylation of Zeta-chain-associated protein kinase 70 (Zap70) and CD3ξ and recruitment of Zap70 and lymphocyte-specific protein tyrosine kinase (Lck) to the immunological synapse. Thus, in the absence of RhoH the subsequent downstream events including phosphorylation of IL2-inducible T-cell kinase (ITK) and complex formation mediated by the linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76) are disrupted. Phosphorylated ITK normally also activates phospholipase C (PLC) which then induces generation of phosphor-inositol-triphosphate (PtdIns) regulating calcium (Ca2+) flux and diacylglycerol (DAG) that activates members of the protein kinase C and RAS guanyl-releasing protein family. These proteins regulate activation of mitogenactivated protein kinases (MAPK) such as JUN amino-terminal kinase, extracellular-signal-regulated kinase 1 and other effectors involved in regulation of gene transcription. Following TCR activation cytoskeleton modifications are also mediated by two downstream pathways that are organized by the LAT-SLP76 complex and thus defective in the absence of RhoH. One pathway depends on degranulation promoting adaptor protein (ADAP), which controls T cell adhesion to the antigen-presenting cell (APC) by up-regulating leukocyte function-associated antigen 1 (LFA1) integrin avidity and the other pathway involves Cdc42, the tyrosine phosphorylated guanine nucleotide exchange factor Vav1, which activates Rac GTPases and controls actin polymerization and TCR clustering through Wiscott-Aldrich syndrome protein (WASP) and the actin nucleating actin related protein (ARP)-complex. Thus, lack of RhoH results in defective TCR signaling, migration and adhesion. Proto-oncogene tyrosine-protein kinase (Fyn), non-catalytic region of tyrosine kinase adaptor protein 1 (Nck), Grb2-like adaptor protein (GADS), growth factor receptor-bound protein 2 (Grb2), Ras guanine nucleotide exchange protein Son of sevenless (Sos), Phosphoinositid-3-Kinase (PI3K) . (Figure adapted from). Figure 1C. Impaired recruitment of Lck to the immunological synapse in Rhoh−/− T cells. CD8+ T cells from wt or Rhoh−/− p14 TCR transgenic mice were conjugated with gp33 peptide-preloaded APC cells (CH.B2 cells) for 5 min. Cells were fixed and stained with anti-Zap70 (green) and anti-Lck (red). The localization of the immune synapse is indicated with a white arrow. T cell-APC conjugates were fixed and stained with TRITC-Phalloidin for detection of F-actin. Differential interference contrast (DIC) images show the antigen-specific T cell-APC conjugates. In the absence of RhoH localization of Zap70 and LCK to the immunological synapse is impaired, which indicates that RhoH is required for activation and recruitment of Zap70 and Lck to the immunological synapse. (Figure from).

Figure 1. Involvement of Rac in phagocytic cell function and RhoH in T cell receptor signaling

Figure 1A. Involvement of Rac in NADPH oxidase activation and superoxide production and cell migration. Inactive GDP and RhoGDP inhibitor(RhoGDI)-bound Rac is located in the cytoplasm. Activation signals mediated by formylmethionyl-leucyl-phenylalanine (fMLP) binding to its G-coupled receptor, results in dissociation of RhoGDI from Rac and subsequent cycling of Rac to the GTP-bound, active form. Together with p40^phox, p47^phox and the p67^phox subunit, activated Rac translocates to the cell or phagosomal membrane to form the active NADPH oxidase complex together with the membrane-bound proteins large glycosylated protein (gp91^phox) and the smaller adaptor protein p22^phox. Subsequent electron transfer from NADPH to molecular oxygen via flavin adenine dinucleotide (FAD) leads to formation of superoxide (O2-) radicals. It is postulated that phosphorylation of p47^phox and p67^phox is mediated by protein kinase C (PKC) and that PKC is also involved in phosphorylation and dissociation of RhoGDI from Rac and thus regulates Rac activation. Activated, GTP-bound Rac also regulates cytoskeleton reorganization via downstream mediators of interaction with F-actin. Lack of Rac therefore disrupts formation of the phagocytic oxidase complex and inhibits migration in phagocytes. (Figure adapted from). Figure 1B. Involvement of RhoH in T cell receptor signaling. The T cell receptor (TCR) complex consists of TCR chains, CD3 and ξ-chain accessory molecules and the co-receptor CD4 or CD8. RhoH is required for phosphorylation of Zeta-chain-associated protein kinase 70 (Zap70) and CD3ξ and recruitment of Zap70 and lymphocyte-specific protein tyrosine kinase (Lck) to the immunological synapse. Thus, in the absence of RhoH the subsequent downstream events including phosphorylation of IL2-inducible T-cell kinase (ITK) and complex formation mediated by the linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76) are disrupted. Phosphorylated ITK normally also activates phospholipase C (PLC) which then induces generation of phosphor-inositol-triphosphate (PtdIns) regulating calcium (Ca2+) flux and diacylglycerol (DAG) that activates members of the protein kinase C and RAS guanyl-releasing protein family. These proteins regulate activation of mitogenactivated protein kinases (MAPK) such as JUN amino-terminal kinase, extracellular-signal-regulated kinase 1 and other effectors involved in regulation of gene transcription. Following TCR activation cytoskeleton modifications are also mediated by two downstream pathways that are organized by the LAT-SLP76 complex and thus defective in the absence of RhoH. One pathway depends on degranulation promoting adaptor protein (ADAP), which controls T cell adhesion to the antigen-presenting cell (APC) by up-regulating leukocyte function-associated antigen 1 (LFA1) integrin avidity and the other pathway involves Cdc42, the tyrosine phosphorylated guanine nucleotide exchange factor Vav1, which activates Rac GTPases and controls actin polymerization and TCR clustering through Wiscott-Aldrich syndrome protein (WASP) and the actin nucleating actin related protein (ARP)-complex. Thus, lack of RhoH results in defective TCR signaling, migration and adhesion. Proto-oncogene tyrosine-protein kinase (Fyn), non-catalytic region of tyrosine kinase adaptor protein 1 (Nck), Grb2-like adaptor protein (GADS), growth factor receptor-bound protein 2 (Grb2), Ras guanine nucleotide exchange protein Son of sevenless (Sos), Phosphoinositid-3-Kinase (PI3K) . (Figure adapted from). Figure 1C. Impaired recruitment of Lck to the immunological synapse in Rhoh−/− T cells. CD8+ T cells from wt or Rhoh−/− p14 TCR transgenic mice were conjugated with gp33 peptide-preloaded APC cells (CH.B2 cells) for 5 min. Cells were fixed and stained with anti-Zap70 (green) and anti-Lck (red). The localization of the immune synapse is indicated with a white arrow. T cell-APC conjugates were fixed and stained with TRITC-Phalloidin for detection of F-actin. Differential interference contrast (DIC) images show the antigen-specific T cell-APC conjugates. In the absence of RhoH localization of Zap70 and LCK to the immunological synapse is impaired, which indicates that RhoH is required for activation and recruitment of Zap70 and Lck to the immunological synapse. (Figure from).

Figure 2. Crosstalk between Rho GTPases

Figure 2A. Crosstalk between GTPases. Members of the RhoGTPase family such as RhoA, RhoG, Rac and Cdc42 appear to have antagonistic effects on cellular functions concerning actin reorganization, gene transcription and protein activation compared to RhoB, RhoD, RhoE and RhoH in various cellular systems. Furthermore it has been demonstrated that members of the Rho GTPase family influence each other directly or via interaction with regulating proteins such as guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) and thus act within an intricate regulatory network. (Figure adapted from). An example for this crosstalk is exhibited in Figure 2B in which absence of RhoH is associated with abnormal accumulation of Rac1 in the cell membrane. (Figure from). Figure 2B. RhoH influences subcellular localization of endogenous Rac in hematopoietic stem cells (HSC): Lin-/c-kit- cells (HSC) were fixed and stained with anti-Rac1 mAB (green), rhodamine-labeled phalloidin (red) and 4,6-diamidino-2-phenylindole (blue). Membrane localization is indicated with white arrows. In the absence of RhoH membrane localization of Rac1 is enhanced, which is associated with increased Rac activity, suggesting a regulatory role of RhoH in Rac activation and subcellular localization. (Figure from).