The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2.

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and ß-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2.

Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.

Herpes simplex virus requires VP11/12 to activate Src family kinase-phosphoinositide 3-kinase-Akt signaling.

We previously showed that the herpes simplex virus 1 (HSV-1) tegument protein VP11/12 activates the lymphocyte-specific Src family kinase (SFK) Lck and is tyrosine phosphorylated in an Lck-dependent manner during T cell infection. We now extend these findings to show that ectopic expression of Lck induces robust tyrosine phosphorylation of VP11/12 in Vero cells, strongly suggesting that VP11/12 participates in an Lck-mediated signaling pathway as a substrate of Lck or a kinase activated by Lck. We sought to elucidate signaling events downstream of VP11/12-SFK interactions. SFKs lie upstream of the canonical phosphoinositide 3-kinase (PI3K)-Akt pathway in signaling emanating from immune receptors, growth factor receptors, and polyomavirus middle T antigen. Here, we show that VP11/12 is required for virus-induced activation of PI3K-Akt signaling in HSV-infected Jurkat T cells and primary fibroblasts. VP11/12 interacts with PI3K or PI3K signaling complexes during infection, suggesting that VP11/12 activates PI3K directly. SFK activity is required for tyrosine phosphorylation of VP11/12, VP11/12-PI3K interactions, and Akt activation in infected fibroblasts, suggesting that SFK-dependent phosphorylation of VP11/12 is required for interactions with downstream signaling effectors. Akt controls many biological functions, including cell survival, cell motility, and translation, but it is currently unclear which Akt targets are modulated by VP11/12 during infection. Although the Akt target mTORC1 is activated during HSV-1 infection, VP11/12 is not required for this effect, implying that one or more additional viral proteins regulate this pathway. Further studies are therefore required to determine which Akt targets and associated biological functions are uniquely modulated by VP11/12.

Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes.

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus.