The Abelson (ABL) family of nonreceptor tyrosine kinases, ABL1 and ABL2, transduces diverse extracellular signals to protein networks that control proliferation, survival, migration, and invasion. ABL1 was first identified as an oncogene required for the development of leukemias initiated by retroviruses or chromosome translocations. The demonstration that small molecule ABL kinase inhibitors effectively treat chronic myeloid leukemia opened the door to the era of targeted cancer therapies. Recent reports have uncovered roles for ABL kinases in solid tumors. Enhanced ABL expression and activation in some solid tumors, together with altered cell polarity, invasion or growth induced by activated ABL kinases, suggest that drugs targeting these kinases may have utility in treating selected solid tumors.

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1␣, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.Podosomes and invadopodia are specialized protrusive structures consisting of a core assembly of F-actin-and actinbinding proteins that form on the ventral surface of migratory and invading cells. These structures are observed in physiological and pathological processes that involve remodeling of the extracellular environment and are found in endothelial cells during extracellular matrix (ECM) 5 degradation (1), transmigrating monocytic cells (2, 3), osteoclasts during bone reabsorption (4), and cancer cells during invasion and metastasis (5). Although podosomes and invadopodia are structurally distinct, they share many common features such as the enrichment of integrins, actin regulatory proteins, matrix metalloproteinases (MMPs), and tyrosine kinases (6 -8).Carcinoma cells utilize invadopodia to degrade the ECM during tumor invasion and metastasis (8). Invadopodia assembly occurs through sequential steps that begin with the assembly of precursor structures containing actin, cortactin, Tsk5, N-WASP, and other actin regulatory proteins, and progress into mature structures with matrix degradation activity (9). Invadopodia were first described in cells transformed with oncogenic v-Src (10), and endogenous Src kinases have been shown to promote podosome/invadopodia formation in response to growth factors and chemokines (1, 11-13). Src phosphorylates several invadopodia components including cortactin, N-WASP, and Tsk5/FISH (14). Cortactin regulates the formation ...

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr 365 in the receptor tail; distal sites were more phosphorylated in some heterologous models. ␤-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nM TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t1 ⁄2 ϳ 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t1 ⁄2 ϳ 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin-and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.The type I thyrotropin-releasing hormone (TRH) 2 receptor is a seven-transmembrane G protein-coupled receptor (GPCR) that is expressed in the anterior pituitary, where it responds to TRH secreted from the hypothalamus, leading to the release of thyroid-stimulating hormone (TSH) from thyrotrophs, which in turn causes release of T 3 and T 4 from the thyroid. Thyroid hormones exert negative feedback at the level of the hypothalamus and pituitary in order to ensure that hormone levels are tightly regulated. Lactotrophs in the anterior pituitary also express TRH receptors and release prolactin after exposure to TRH. In addition to the anterior pituitary, the TRH receptor is expressed in various areas of the central nervous system. The TRH receptor signals via G␣ q/11 , which activates phospholipase C␤ and leads to the generation of inositol 1,4,5-trisphosphate and diacylglycerol, the release of calcium from the endoplasmic reticulum, and the activation of protein kinase C (PKC).GPCRs can be phosphorylated by second messenger-activated kinases, such as PKC, or by GPCR kinases (GRKs), which preferentially recognize the agonist-occupied receptor conformation (1, 2). Like other GPCRs, the TRH receptor is phosphorylated after binding to agonist (3, 4), whi...

Phagocytosis of antibody-coated pathogens is mediated through Fcγ receptors (FcγRs) which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages, and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the Syk kinase. Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X) 5-6 F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the ␤2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.The type 1 TRH receptor is a GPCR expressed in the anterior pituitary gland. In response to TRH, the receptor activates G q/11 , which in turn stimulates phospholipase C. The resulting increases in inositol trisphosphate and diacylglycerol cause rapid release of intracellular calcium and activation of protein kinase C. Like many GPCRs, the TRH receptor contains a canonical NPXXY at the end of transmembrane 7 in a NPXXY(X) 5-6 F(R/K) motif (Mirzadegan et al., 2003;Okuno et al., 2005;Swift et al., 2006) and is predicted to form an amphipathic eighth helix with a positively charged face ending at a downstream pair of cysteine residues, where palmitoylation occurs (Du et al., 2005).When TRH binds, the receptor undergoes rapid and quantitative phosphorylation at multiple sites in the cytoplasmic tail Hinkle, 2005, 2008;Jones et al., 2007). Agonist-stimulated phosphorylation of the receptor drives arrestin binding and arrestin-dependent receptor desensitization and internalization. The receptor tail is not detectably phosphorylated before activation.Agonist-dependent phosphorylation of GPCRs is usually carried out by either GRKs that recognize the activated state of the receptor or downstream kinases that are activated by receptor signaling. Th...