Complex kinase requirements forChlamydia trachomatisTarp phosphorylation

Abstract

Chlamydia trachomatis translocates the effector protein Tarp (translocated actin-recruiting phosphoprotein) into the host cell cytoplasm where it is quickly tyrosine phosphorylated. Abl and Src kinases have been implicated in Tarp phosphorylation; however, we observed that the situation is more complex. Chemical inhibition of Src family kinases confirmed a role for these kinases in Tarp phosphorylation. Infection of Src, Yes, Fyn (SYF)-deficient cells showed a dampened, but incompletely blocked, Tarp phosphorylation. Inhibition of Abl in an SYF background still did not completely block Tarp phosphorylation. Consequently, we tested additional kinases and found that Syk, but not Btk or Jak2, is a potent kinase of Tarp in vitro. Inhibition of Syk in an SYF background further blocked Tarp phosphorylation. Under these conditions, inclusion formation still proceeded normally. These data reveal a highly promiscuous substrate property of Tarp and set the stage for further functional characterization of Tarp phosphorylation during host cell infection.

Chlamydia trachomatis Tarp Harbors Distinct G and F Actin Binding Domains That Bundle Actin Filaments

“…C. trachomatis Tarp orthologs are phosphorylated in the mammalian host by a variety of tyrosine kinases, including Src family kinases, Abl, and Syk (27,28). SH2 domains found in the host proteome specifically associate with phosphotyrosine-containing polypeptides and are potential Tarp-interacting proteins (29).…”

Tarp regulates earlyChlamydia-induced host cell survival through interactions with the human adaptor protein SHC1

“…Genes of groups 2 and 4 show an additional regulation by C. trachomatis infection independent of SHC1, which remains after SHC1 knockdown in infected cells. Jewett et al, 2008;Mehlitz et al, 2008). Consequently, the interaction between nonphosphorylated Tarp and ABL, leading to subsequent activation of ABL, may constitute an initial trigger for Tarp phosphorylation (see model in Fig.…”

Chlamydial Intracellular Survival Strategies

“…At least one early secreted effector, TARP, contributes to bacterial internalization by its ability to directly nucleate actin polymerization through a WH2 actin-binding domain mimic (Jewett et al 2006) and by recruiting the guanine nucleotide exchange factors (GEFs) Sos1 and Vav2, which activate Rac1 and signal to the actin machinery (Carabeo et al 2007;Lane et al 2008). This latter mechanism is only relevant in chlamydial species inwhich the polymorphic TARP gene (Clifton et al 2005) can be targeted for tyrosine phosphorylation by Abl, Src, and Syk kinases (Elwell et al 2008;Jewett et al 2008;Lane et al 2008;Mehlitz et al 2008Mehlitz et al , 2010. Microinjection of cells with antibodies directed to the actin-binding domain of TARP before infection significantly reduces bacterial invasion, providing the most conclusive support for a direct role of TARP in mediating bacterial entry (Jewett et al 2010).…”