SecPH partially inhibits cofilin phosphorylation by LIMK2, but not by LIMK1.

<p><i>A</i>. <i>Actin cytoskeleton organisation</i>. HeLa cells were cotransfected with pcDNA3, LIMK2 and SecPH or its parental empty plasmid, p3XFlag. Cells were fixed and stained with phalloidin, or anti-HA or anti-flag antibodies. <i>B. Inhibition of LIMK2 cofilin phosphorylation by SecPH.</i> Cells were cotransfected with LIMK2 and SecPH or Galectin-3 (a non-specific control protein). Immunoprecipitated HA-LIMK2 and GST-cofilin were used in the kinase assay. The kinase activity on cofilin of immunoprecipitated HA-LIMK2 from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. Statistical significance was determined relative to control using one-way ANOVA (* p<0.05). The HA-immunoprecipitates were also submitted to HA-immunoblotting and to coomassie blue staining. Lysates were submitted to flag-immunoblotting. <i>C. Dose dependent inhibition of LIMK2 cofilin phosphorylation by SecPH.</i> Cells were transfected with LIMK2 and either SecPH or Galectin-3. SecPH and Galectin-3 cell lysates were immunoprecipitated with anti-flag beads, beads were then eluted flag peptide. Immunoprecipitated HA-LIMK2 and GST-cofilin were used for kinase assay and were incubated with increasing amount of immunoprecipitated SecPH or Galectin-3 (0, 6, 12, 18 ul respectively). The kinase activity on cofilin of immunoprecipitated HA-LIMK2 with no addition of immunoprecipitated SecPH or Galectin-3 was taken as 1.0. Each value represents the mean ± SE of four independent experiments. Statistical significance was determined relative to control using one-way ANOVA (*** p<0.0001). Immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining. <i>D. SecPH does not inhibit cofilin phosphorylation by LIMK1.</i> Cells were transfected either with SecPH or with LIMK1. SecPH cell lysates were immunoprecipitated with anti-flag beads, beads were then eluted with flag peptide. Immunoprecipitated HA-LIMK1 was used for kinase assay and was incubated with increasing amount of immunoprecipitated SecPH (0, 6, 12, 18 ul respectively). The kinase activity on cofilin of immunoprecipitated HA-LIMK1 with no addition of immunoprecipitated SecPH was taken as 1.0. Each value represents the mean ± SE of two independent experiments. Immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining.</p

Schematic representation of our findings: A molecular connection between neurofibromin and the Rho/ROCK/LIMK2/cofilin pathway.

<p><i>A.</i> Upon Rho activation <i>via</i> binding to its RBD (Rho Binding domain), ROCK activates LIMK2 by phosphorylation at its Thr505. Activated LIMK2 will then phosphorylate cofilin on its Ser3, resulting in its inhibition. An invasive phenotype is then observed with accumulation of actin stress fibers. <i>B. SecPH, a new inhibitor of this Rho/ROCK/LIMK2/cofilin pathway</i>. By interacting with LIMK2, SecPH prevents ROCK activation of LIMK2. Our data raises two possible hypotheses for this inhibition of ROCK activation of LIMK2 by SecPH: (1) by steric hindrance, SecPH hides Thr505 of LIMK2 from ROCK accessibility, (2) the PH domain of SecPH substitutes to the PH domain of ROCK by inhibiting the kinase activity of ROCK; this inhibition would specifically occur when ROCK and SecPH are simultaneously bound to LIMK2.</p

Mechanism of SecPH inhibition of LIMK2 kinase activity.

<p><i>A. SecPH does not prevent LIMK2 from interacting with ROCK1</i>. Cells were cotransfected with HA-LIMK2, ROCK1 and SecPH or its empty parental plasmid. Lysates and anti-HA immunoprecipitates were subjected to immunoblotting. <i>B. SecPH affects LIMK2 phosphorylation.</i> Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone-0047283-g002" target="_blank">Figure 2B</a>. Statistical significance was determined relative to control using one-way ANOVA (*** p<0.0001). <i>C. Inhibition of LIMK2 cofilin phosphorylation by SecPH in the presence of ROCK1.</i> Cells were cotransfected with ROCK1, LIMK2 and SecPH or Galectin-3 (a non-specific control protein). Immunoprecipitated HA-LIMK2 and GST-cofilin were used for the kinase assay. The kinase activity on cofilin of immunoprecipitated HA-LIMK2 from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. The HA-immunoprecipitates were also submitted to immunoblotting and to coomassie blue staining. Lysates were also submitted to flag-immunoblotting. <i>D. SecPH affects LIMK2 T505 phosphorylation by ROCK1.</i> Cells were cotransfected with either LIMK2-TA or LIMK2-WT and SecPH or Galectin-3 (a non-specific control protein). Lysates were subjected to immunoblotting.</p

SecPH inhibition of cofilin phosphorylation by LIMK2 requires ROCK activation of LIMK2.

<p><i>A. SecPH interacts with LIMK2 whatever its activation state.</i> Cells were cotransfected with SecPH and either LIMK2-WT, or LIMK2-TA or LIMK2-TEE. Lysates and anti-flag immunoprecipitates were subjected to immunoblotting. <i>B. Nf1 interacts with LIMK2 whatever its activation state.</i> Cells were transfected with either LIMK2-WT, or LIMK2-TA or LIMK2-TEE. Lysates and anti-HA immunoprecipitates were subjected to immunoblotting. <i>C. SecPH is unable to modulate cofilin phosphorylation by LIMK2-T505 mutants</i>. Cells were cotransfected with either LIMK2-WT, or LIMK2-TA or LIMK2-TEE and SecPH or its parental empty plasmid. Immunoprecipitated HA-LIMK2 and GST-cofilin were used in the kinase assay. Anti-HA immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining.</p

SecPH affects ROCK kinase activity specifically with respect to LIMK2.

<p><i>A. MLC phosphorylation is not affected by SecPH.</i> Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone-0047283-g002" target="_blank">Figure 2B</a>, except 0.5 µg of MLC were added in the kinase reaction mixture. Each value represents the mean ± SE (standard error) of two independent experiments. B. <i>MLC phosphorylation by ROCK-1 is not affected by SecPH.</i> Cells were cotransfected with ROCK1 and SecPH or Galectin-3. Immunoprecipitated c-Myc-ROCK1 was used for the kinase assay in the presence of recombinant LIMK2 or MLC (0.5 µg each). The kinase activity on LIMK2/MLC of immunoprecipitated c-Myc-ROCK from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. Immunoprecipitates were also submitted to c-Myc-immunoblotting and lysates to flag-immunoblotting.</p

Interaction between LIMK2 and the SecPH domain of Nf1.

<p><i>A. Diagram of Nf1.</i> GRD (GAP related domain) responsible for the main known function of Nf1 is depicted as well as SecPH, the region used for the two-hybrid screening. <i>B. Interaction revealed by the two-hybrid screening.</i> L40 cells transformed with pBTM116-TBD were mated with Y187 cells transformed with the empty plasmid pACT2 (as a negative control) or pACT2-TFS1 (as a positive control, as demonstrated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone.0047283-Chautard1" target="_blank">[32]</a>). L40 cells transformed with pBTM116-SecPH were mated with Y187 cells transformed with pACT2-LIMK1 or pACT2-LIMK2. After mating on YPD, the resultant diploids were selected on a SD-LW medium. The interaction between the LexA fusion proteins encoded by the pACT2 plasmids and the Gal4 fusion proteins encoded by pBTM116 plasmids was tested by checking the growth of diploids on a SD-LWH media containing 3AT (1 mM) and their ability to cleave X-gal (1 mM) thereby attesting the production of β-galactosidase. <i>C. Interaction in HEK-293 transfected cells.</i> HEK-293 cells were cotransfected with either HA-LIMK2 or HA-LIMK1 and flag-SecPH or its parental empty plasmid (p3XFlag). Cell lysates and anti-flag immunoprecipitation eluates were analyzed by immunobloting. <i>D. Immunoprecipitated LIMK2 interacts with recombinant 6His-SecPH.</i> HEK-293 cells were transfected with HA-LIMK2 or its parental empty plasmid, pcDNA3. The corresponding cell lysates were immunoprecipitated with anti-HA beads. Beads were then incubated with 6His-SecPH in lysis buffer. Anti-HA immunoprecipitates were analyzed by immunobloting. <i>E. Transfected LIMK2 interacts with endogenous Nf1.</i> Cells were transfected with HA-LIMK2 or its parental empty plasmid, pcDNA3. Lysates and anti-HA immunoprecipitates were analyzed by immunobloting. <i>F. Endogenous LIMK2 interacts with endogenous Nf1</i> Anti-Nf1 immunoprecipitates from HEK-293 were analyzed by immunobloting. <i>G. Domains of LIMK2 involved in its interaction with SecPH</i>. Top. Schematic diagram of LIMK2 and its various fragments designed for this study. Bottom. Cells were cotransfected with SecPH and one of the domains of LIMK2. Lysates and anti-flag immunoprecipitates were analyzed by immunobloting.</p

List of plasmids used in this study.

<p>List of plasmids used in this study.</p

Design, Synthesis, and Biological Activity of Pyridopyrimidine Scaffolds as Novel PI3K/mTOR Dual Inhibitors

The design, synthesis, and screening of dual PI3K/mTOR inhibitors that gave nanomolar enzymatic and cellular activities on both targets with an acceptable kinase selectivity profile are described. A docking study was performed to understand the binding mode of the compounds and to explain the differences in biological activity. In addition, cellular effects of the best dual inhibitors were determined on six cancer cell lines and compared to those on a healthy diploid cell line for cellular cytotoxicity. Two compounds are highly potent on cancer cells in the submicromolar range without any toxicity on healthy cells. A more detailed analysis of the cellular effect of these PI3K/mTOR dual inhibitors demonstrated that they induce G1-phase cell cycle arrest in breast cancer cells and trigger apoptosis. These compounds show an interesting kinase profile as dual PI3K/mTOR tool compounds or as a chemical series for further optimization to progress into in vivo experiments