Cytoskeletal rearrangement and Src and PI-3K-dependent Akt activation control GABABR-mediated chemotaxis

The goal of this study was to define signaling mechanisms regulating GABABR-mediated chemotaxis and cytoskeletal rearrangement. In a proteomic study we identified serine/threonine kinase Akt, tyrosine kinases Src and Pyk2, microtubule regulator kinesin and microtubule affinity-regulating kinase (MARK) co-immunoprecipitating with GABABR. To define the contributions of these candidate signaling events in GABABR-mediated chemotaxis, we used rat basophilic leukemic cells (RBL-2H3 cells) stably transfected with human GABAB1b and GABAB2 receptors. The GABABR agonist baclofen induced Akt phosphorylation and chemotaxis by binding to its specific GABABR since pretreatment of cells with CGP52432, a GABABR antagonist, blocked such effects. Moreover, baclofen induced Akt phosphorylation was shown to be dependent upon PI-3K and Src kinases. Baclofen failed to stimulate actin polymerization in suspended RBL cells unless exposed to a baclofen gradient. However, baclofen stimulated both actin and tubulin polymerization in adherent RBL-GABABR cells. Blockade of actin and tubulin polymerization by treatment of cells with cytochalasin D or nocodazole respectively, abolished baclofen-mediated chemotaxis. Furthermore, baclofen stimulated Pyk2 and STAT3 phosphorylation, both known regulators of cell migration. In conclusion, GABABR stimulation promotes chemotaxis in RBL cells which is dependent on signaling via PI3-K/Akt, Src kinases and on rearrangement of both microtubules and actin cytoskeleton. These data define mechanisms of GABABR-mediated chemotaxis which may potentially be used to therapeutically regulate cellular response to injury and disease.