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Sprouty-4 negatively regulates cell spreading by inhibiting the kinase activity of testicular protein kinase.

ABSTRACT: TESK1 (testicular protein kinase 1) is a serine/threonine kinase that phosphorylates cofilin and plays a critical role in integrin-mediated actin cytoskeletal reorganization and cell spreading. We previously showed that TESK1 interacts with Sprouty-4 (referred to as Spry4), an inhibitor of growth factor-induced Ras/MAP (mitogen-activated protein) kinase signalling, but the functional role of this interaction has remained unknown. In the present study, we show that Spry4 inhibits the kinase activity of TESK1 by binding to it through the C-terminal cysteine-rich region. Expression of Spry4 in cultured cells suppressed integrin-mediated cell spreading, and TESK1 reversed the inhibitory effect of Spry4 on cell spreading. Furthermore, Spry4 suppressed integrin- and TESK1-mediated cofilin phosphorylation during the spreading of cells on laminin. These findings suggest that Spry4 suppresses cell spreading by inhibiting the kinase activity of TESK1. Although tyrosine phosphorylation is required for the inhibitory activity of Spry4 on a Ras/MAP kinase pathway, mutation of the corresponding tyrosine residue (Tyr-75 in human Spry4) to an alanine had no apparent effect on its inhibitory actions on TESK1 activity and cell spreading, which suggests a novel cellular function of Spry to regulate the actin cytoskeleton, independent of its inhibitory activity on the Ras/MAP kinase signalling.

SUBMITTER: Tsumura Y

PROVIDER: S-EPMC1134992 | biostudies-other | 2005 May

REPOSITORIES: biostudies-other

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Project description:The phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of beta interferon (IFN-?) induced by Sendai virus and poly(I·C), while knockdown of Cdc25A enhances the transcription of IFN-? stimulated by RNA virus infection. The inhibitory effect of Cdc25A on the antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-?. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A upregulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits the antiviral immune response by reducing the active form of TBK1. Using herpes simplex virus 1 (HSV-1) infection, an IFN-? reporter assay, and reverse transcription-quantitative PCR (RT-qPCR), we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-?. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that Cdc25A can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates the antiviral immune response by inhibiting TBK1 activity.IMPORTANCE The RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism for balancing the RLR signaling pathway in host cells is not well understood. We found that the phosphatase Cdc25A negatively regulates the RNA virus-induced innate immune response. Our studies indicate that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-? transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-?. Taken together, our findings uncover a novel function and mechanism for Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity and demonstrate its role in maintaining host cell homeostasis following viral infection.

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Sprouty-4 negatively regulates cell spreading by inhibiting the kinase activity of testicular protein kinase.

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