Rho family GTPases and their regulators in lymphocytes

Abstract

Rho family GTPases, and the proteins that regulate them, have important roles in many cellular processes, including cell division, survival, migration and adhesion. Although most of our understanding of these proteins has come from studies using cell lines, more recent gene targeting studies in mice are providing insights into the in vivo function of these proteins. Here we review recent progress revealing crucial roles for these proteins in lymphocyte development, activation, differentiation and migration. The emerging picture shows that Rho family GTPases transduce signals from receptors for antigens, chemokines and cytokines, as well as adhesion molecules and pattern recognition receptors, and that they function as focal points for crosstalk between different signalling pathways.

Figure 1

Mouse Rho GTPases. Phylogenetic tree of 23 mouse Rho GTPases, showing how they cluster into different subfamilies, for example the Rac, RhoA/RhoB/RhoC and Rnd subfamilies. We note that it has been proposed that MIRO1, MIRO2 and RHOBTB3 do not belong to the Rho GTPase family.

Figure 2

Regulation of Rho GTPases. This figure shows a generic Rho GTPase anchored to the membrane with a prenyl group near the C-terminus (zigzag line). The GTPase binds either GDP or GTP. Guanine nucleotide-exchange factors (GEFs) catalyse the release of GDP from the GTPase, allowing GTP to bind. GTPase-activating proteins (GAPs) increase the intrinsic GTPase activity of the Rho proteins, causing GTP to be hydrolysed to GDP and phosphate (P_i). GEFs and GAPs are often constitutively or inducibly associated with membranes. GDP-bound Rho proteins can be sequestered by Rho guanine nucleotide dissociation inhibitors (GDIs), which bind to the lipid modification and thereby inhibit membrane binding of the GTPase. GTP-bound Rho proteins transduce signals by binding to effector proteins.

Figure 3

Role of Rho GTPases and their regulators in B cell development and function. The earliest B cell progenitors in the bone marrow, pro-B cells, rearrange immunoglobulin heavy chain genes. If they successfully generate a heavy chain, this assembles into the pre-B cell receptor (pre-BCR). Signals from the pre-BCR allow cells to differentiate into pre-B cells and proliferate. Pre-B cells rearrange light chain genes, and if this process is successful, the light chain pairs with the heavy chain to generate cell surface-bound BCR in the form of IgM. These cells, known as immature B cells, exit the bone marrow and migrate through the circulation to the red pulp of the spleen, where we have recently described them as transitional type 0 (T0) cells (R. Henderson and V. Tybulewicz, unpublished observations). Chemokine-induced migration of T0 cells into the white pulp is accompanied by maturation into transitional type 1 (T1) and then type 2 (T2) cells, and finally into either follicular or marginal zone (MZ) B cells. Follicular B cells recirculate between secondary lymphoid organs, including spleen, lymph node, Peyer’s patches and bone marrow. B1 cells, a distinct lineage of B cells, which predominate in the peritoneal and pleural cavities, are derived from foetal progenitors (not shown). Activation of naïve follicular B cells (thick arrow) results in their movement into germinal centres (GCs) where, as GC B cells, with T cell help, they undergo somatic hypermutation, affinity maturation and class switching. Eventually the cells mature into antibody-secreting plasma cells and memory B cells. Points where roles for Rho GTPases or their regulators have been identified are indicated, and discussed more fully in the main text.

Figure 4

Signal transduction from the B cell receptor. Binding of antigen to the B cell receptor (BCR) leads to activation of the tyrosine kinase LYN, phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of Igα and Igβ, recruitment and activation of the tyrosine kinase SYK (spleen tyrosine kinase), phosphorylation of the adaptor protein BLNK (B cell linker) and assembly of a complex including BTK (Bruton’s tyrosine kinase), VAV1 and PLCγ2 (phospholipase Cγ2). Activation of PLCγ2 leads to hydrolysis of the lipid phosphatidylinositol-4,5-bisphosphate (Ptd(4,5)InsP₂) to the second messengers inositol-3,4,5-trisphosphate (InsP₃) and diacylglycerol (DAG). Ptd(4,5)InsP₂ is synthesized by sequential phosphorylation of phosphatidylinositol (PtdIns) to phosphatidylinositol-4-phosphate (Ptd(4)InsP) and then to Ptd(4,5)InsP₂ by phosphatidylinositol-4-kinase (PI4K) and phosphatidylinositol-4-phosphate-5-kinase (PIP5K) respectively. Both of these latter kinases are activated by RHOA. VAV1 may contribute to the activation of PLCγ2 through an adaptor function, and, via RAC1 lead to increased synthesis of PtdIns(3,4,5)P₃, potentially by activating phosphatidylinositide 3-kinase (PI3K). Increased levels of InsP₃ lead to an increase in intracellular Ca²⁺ and eventually to activation of nuclear factor of activated T cells (NFAT). DAG activates the RAS exchange factor RASGRP, leading to activation of RAS and the RAF, MEK, ERK kinase cascade. Ca²⁺ and DAG may also contribute to activation of nuclear factor-kB (NF-κB) via PKCβ. PtdIns(3,4,5)P₃ leads to activation of the kinase AKT which signals cell survival through inhibition of the FOXO transcription factors. Signalling from the BCR also activates αPIX, and hence RAC1 and PAK.

Figure 5

Signal transduction from G-protein-coupled receptors and integrins. Receptors for sphingosine 1-phosphate (S1P) and thromboxane A₂ (TXA₂) are members of the G-protein-coupled receptor (GPCR) family, and signal through Gα12 and Gα13 proteins to LSC and RHOA activation. LSC inhibits Gα12/13 function through its RGS domain. Chemokine receptors signal through Gαi2 to DOCK2 and other guanine nucleotide-exchange factors (GEFs) leading to the activation of RAC1 and RHOA. These GTPases transduce signals to integrin activation through phospholipase D (PLD) and PIP5K1C. Integrin signalling is inhibited by RHOH through an unknown mechanism. Integrin signalling leads to the activation of the αPIX GEF and thus the activation of RAC1 and PAK. In myeloid cells, integrin signalling also leads to activation of SRC family kinases which phosphorylate immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor subunits, such as Fc receptor γ-chain (FcRγ), leading to recruitment and activation of spleen tyrosine kinase (SYK). SYK in turn phosphorylates SH2 domain-containing leukocyte protein of 76 kDa (SLP76) leading to assembly of a complex including PLCγ1, NCK and VAV1, and hence to activation of RAC1 and CDC42. It is not known if this latter pathway operates in lymphocytes.

Figure 6

Role of Rho GTPases and their regulators in T cell development and function. The earliest T cell progenitors, CD4^-CD8^- double negative (DN) thymocytes, rearrange T cell receptor β (TCRβ) chain genes. If they successfully generate a TCRβ chain, this assembles into the pre-TCR. Signals from the pre-TCR allow cells to differentiate into CD4⁺CD8⁺ double positive (DP) cells and proliferate. DP thymocytes rearrange TCRα chain genes, and if this process is successful, the TCRα chain pairs with TCRβto generate cell-surface-bound TCR. Signalling from the TCR on DP cells results in positive or negative selection depending on avidity of binding to self-peptide MHC complexes. DP cells with a MHC class I-restricted TCR differentiate into CD4^-CD8⁺ single positive (CD8 SP) cells, whereas MHC class II-restricted cells are directed into CD4⁺CD8^- single positive (CD4 SP) cells. SP thymocytes exit the thymus, migrate through the blood to secondary lymphoid organs, and become either CD4⁺ or CD8⁺ T cells. Activation of naïve CD4⁺ or CD8⁺ T cells (thick arrow) results in their proliferation and differentiation. CD4⁺ T cells can become T helper 1 (T_h1), T_h2 or T_h17 cells, T regulatory (T_reg) cells or memory T cells. CD8⁺ T cells differentiate into cytotoxic T lymphocytes (CTL) and memory T cells. Points where roles for Rho GTPases or their regulators have been identified are indicated and discussed more fully in the main text.

Figure 7

Signal transduction from the T cell receptor. Binding of peptide:MHC complex to the T cell receptor (TCR) leads to activation of the tyrosine kinases LCK and ZAP70 (ζ-chain-associated protein kinase of 70 kDa), phosphorylation of the adaptor protein LAT (linker for activation of T cells) and assembly of a complex including the adaptors GADS, SLP76 and NCK, the ITK and PAK kinases, VAV1 and PLCγ1. Activation of PLCγ1 leads to hydrolysis of PIP2 to the second messengers InsP3 and DAG. VAV1 may contribute to the activation of PLCγ2 through an adaptor function, and, via RAC1 to increased synthesis of PIP3, potentially by activating phosphatidylinositol-3-kinase (PI3K). Increased levels of InsP₃ lead to a rise in intracellular Ca²⁺ and eventually to activation of the NFAT transcription factor. DAG activates the Ras exchange factor RASGRP, leading to activation of RAS and the RAF, MEK, ERK kinase cascade. DAG may also contribute to activation of NFκB via PKCθ. PIP₃ leads to activation of the AKT kinase which signals cell survival via inhibition of the FOXO1 transcription factor. TCR signalling via IBP leads to CDC42 activation and increased intracellular Ca²⁺.