Control Of G Protein Signaling: Role Of The RGSs

National Institute Of Allergy And Infectious Diseases

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Abstract

We established methodology to assess F-actin and myosin IIA dynamics during B cell and neutrophil migration. Using a flow-based assay, we examined the chemoattractant mediated signaling pathway that induced F-actin formation in resting B cells. F-actin levels increased within 5 seconds and peaked at 20 seconds after CXCL13 exposure. F-actin formation depended upon increases in both branched and linear F-actin, Galpha i protein nucleotide exchange, Dock2, and Rac activation. Inhibiting Erk or Src activation, or Myosin IIA reduced, but did not eliminate CXCL13 induced F-actin. Inhibiting Gbeta/gamma signaling slightly reduced the induction of F-actin, while inhibition of BTK or RhoA had no effect. Activated Gi recruited Elmo1 to the plasma membrane. These studies support a prominent role for a Galpha i/Elmo1/Dock2/Rac/Wave regulatory complex signaling pathway in the initial F-actin response of B cells to CXCL13. Using ICAM-1 and chemoattractant coated imaging chambers along with an agarose overlay, we imaged the motility of LifeAct GFP and Myosin IIA-GFP expressing B-lymphocytes and neutrophils. In the under agarose assays the neutrophils migrated much more vigorously than did the B cells, however, a 6 h exposure to LPS significantly improved B cell migration. In both B cells and neutrophils F-actin accumulated at the leading cell edge and in the pseudopods of the migrating cells. Inactivating Gi nucleotide exchange markedly impaired the motility of both the nave and 6 h LPS activated B cells. Myosin IIA weakly accumulated at the leading edge and strongly in the pseudopods. Myosin IIA also accumulated on the lateral edge of turning cell on the side opposite the direction of the turn. Intravital imaging of myosin IIA-GFP B cells confirmed the dynamic behavior of Myosin-IIA during transendothelial and interstitial migration of B cells in vivo. These studies are extending of the signaling pathways and molecules that control B cell movement in vivo. Lymphocyte Function-Associated Antigen 1 (LFA-1) binds Intercellular Adhesion Molecule 1 and 2 (ICAM-1 and ICAM-2) expressed on endothelial cells and antigen presenting cells, and helps coordinate T cell migration, adhesion, and activation. LFA-1 is activated during chemokine and TCR receptor engagement through inside-out signaling via T cell and chemoattractant receptor mediated phosphoinositide 3-kinase (PI3K) activation, and its product PI(3,4,5)P3 (PIP3). To evaluate roles of PIP3-binding proteins in LFA-1 activation, we designed a retroviral library encoding CRISPR/Guide RNAs targeting known and potential PIP3-binding proteins. Using this library to screen for effects on ICAM-1-binding of Cas9-expressing primary mouse T cells, we identified multiple proteins regulating LFA-1-mediated adhesion, including the RAP1/RAS GTPase-activating protein, RASA3. We found RASA3 to be a critical negative regulator of LFA-1 activation that is inhibited by PI3K activity. Imaging experiments identified a strong co-localization between RASA3 and Gi, the major transducer of chemoattractant receptor signaling and RASA3. RASA3 overexpression in HeLa cells caused a marked increase in cytoneme formation. T cells lacking RASA3 exhibited elevated ICAM-1-binding associated with increased RAP1 activation, defective lymph node entry and egress, and impaired help for T-dependent immunization. Our results have uncovered a critical role for RASA3 as a PI3K-regulated inhibitor of T cell adhesion and migration that is required for T cell homeostasis and function (this study is an ongoing collaboration with Pam Schwartzberg's laboratory). We have acquired Rasa3fl/fl mice and have begun to breed these mice with mb1-Cre mice. This will allow deletion of Rasa3 in B lymphocytes and allow assessment of the importance of RASA3 in B cells. Ligand-engaged chemokine receptors trigger heterotrimeric G-protein i subunit nucleotide exchange that stimulates cytoskeletal reorganization and cell polarity changes. To better understand the responsible signaling events, we focused on early F-actin changes following murine splenic B cell CXCR5 engagement. Within 10 seconds three-dimensional lamellar like pseudopods and F-actin rich ridges appeared. The transient F-actin increase depended upon Gi2/3-signaling, the PI-3 kinase/AKT pathway, ERK activation, phospholipase C activity, and Dock2 mediated Rac1/2 activation. AKT substrate and pT58 WNK1 immunoblotting identified WNK1 (with no lysine kinase 1) as a potential early AKT effector. Verifying its importance, treating B cells with specific WNK inhibitors reduced pAKT and pERK activation, disrupted F-actin dynamics, and impaired B cell polarity, motility, and chemotaxis. CRISPR/Cas9 gene editing of WNK1 in a murine B cell line confirmed the inhibitor data and suggested that WNK1 contributes to B cell proliferation. A one-time administration of a WNK inhibitor to mice transiently reduced lymph node B cell motility and polarity in vivo. These results indicate that WNK1 signaling maintains B cell responsiveness to chemokines and suggests that pharmacological inhibition of WNK1 may have untoward effects on humoral immunity. The serine/threonine With-No-Lysine (WNK) kinase family function in blood pressure control, electrolyte homeostasis, and cellular osmoregulation. These kinases and their downstream effectors are considered promising therapeutic targets in hypertension and stroke. However, the role of WNK kinases in immune cells remain poorly understood. We investigated how WNK kinase inhibition affects natural killer (NK) cell physiology. Small-molecule WNK kinase inhibition with WNK463 or WNK-IN-11 dramatically decreased IL-2-activated NK-cell volume, motility and cytolytic activity. Treatment of NK cells with these inhibitors induced autophagy by activating AMPK and inhibiting mTOR signaling. Moreover, WNK kinase inhibition increased phosphorylation of Akt and c-Myc by misaligning activity of activating kinases and inhibitory phosphatases. Treatment of tumor-bearing mice with WNK463 impaired tumor metastasis control by adoptively transferred NK cells. Thus, the catalytic activity of WNK kinases has a critical role of multiple aspect of NK cell physiology and their pharmacologic inhibition negatively impacts NK cell function.