Proteomic Screen for Cellular Targets of the Vaccinia Virus F10 Protein Kinase Reveals that Phosphorylation of mDia Regulates Stress Fiber Formation.
Vaccinia virus, a complex dsDNA virus, is unusual in replicating exclusively within the cytoplasm of infected cells. Although this prototypic poxvirus encodes >200 proteins utilized during infection, a significant role for host proteins and cellular architecture is increasingly evident. The viral B1 kinase and H1 phosphatase are known to target cellular proteins as well as viral substrates, but little is known about the cellular substrates of the F10 kinase. F10 is essential for virion morphogenesis, beginning with the poorly understood process of diversion of membranes from the ER for the purpose of virion membrane biogenesis. To better understand the function of F10, we generated a cell line that carries a single, inducible F10 transgene. Using uninduced and induced cells, we performedstableisotopelabeling ofamino acids incell culture (SILAC) coupled with phosphopeptide analysis to identify cellular targets of F10-mediated phosphorylation. We identified 27 proteins that showed statistically significant changes in phosphorylation upon the expression of the F10 kinase: 18 proteins showed an increase in phosphorylation whereas 9 proteins showed a decrease in phosphorylation. These proteins participate in several distinct cellular processes including cytoskeleton dynamics, membrane trafficking and cellular metabolism. One of the proteins with the greatest change in phosphorylation was mDia, a member of the formin family of cytoskeleton regulators; F10 induction led to increased phosphorylation on Ser22Induction of F10 induced a statistically significant decrease in the percentage of cells with actin stress fibers; however, this change was abrogated when an mDia Ser22Ala variant was expressed. Moreover, expression of a Ser22Asp variant leads to a reduction of stress fibers even in cells not expressing F10. In sum, we present the first unbiased screen for cellular targets of F10-mediated phosphorylation, and in so doing describe a heretofore unknown mechanism for regulating stress fiber formation through phosphorylation of mDia. Data are available via ProteomeXchange with identifier PXD005246.
From the ‡Departments of Biochemistry & Molecular Biology and Microbiology & Immunology, and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina.
Matthew D
MD
Greseth
§Department of Microbiology & Molecular Genetics and the Biotechnology & Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin.
Dominique C
DC
Carter
§Department of Microbiology & Molecular Genetics and the Biotechnology & Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin.
Scott S
SS
Terhune
From the ‡Departments of Biochemistry & Molecular Biology and Microbiology & Immunology, and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina; traktman@musc.edu.
Paula
0000-0002-6517-0225
P
Traktman
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D006801:Humans; D007553:Isotope Labeling; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D060066:Protein Interaction Maps; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012694:Serine / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism*; D014616:Vaccinia virus / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D010750:Phosphoproteins; D014764:Viral Proteins; D012694:Serine; C095546:protein kinase 2, Vaccinia virus; D017346:Protein Serine-Threonine Kinases