A New Dimension to Ras Function: A Novel Role for Nucleotide-Free Ras in Class II Phosphatidylinositol 3-Kinase Beta (PI3KC2 beta) Regulation

The intersectin 1 (ITSN1) scaffold stimulates Ras activation on endocytic vesicles without activating classic Ras effectors. The identification of Class II phosphatidylinositol 3-kinase beta, PI3KC2 beta, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2 beta suggests a role for Ras in PI3KC2 beta activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2 beta activity. PI3KC2 beta preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2 beta interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2 beta. Using purified proteins, we demonstrate that the PI3KC2 beta-RBD directly binds nucleotide-free Ras in vitro and that this interaction is not disrupted by nucleotide addition. Finally, nucleotide-free Ras but not GTP-loaded Ras inhibits PI3KC2 beta lipid kinase activity in vitro. Our findings indicate that PI3KC2 beta interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2 beta stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2 beta mutually inhibit one another

Mutations in the effector region of Ras disrupt PI3KC2β binding.

<p>(A) Point mutations in the effector region of Ras12V disrupt interactions with specific Ras targets. (B) VC-tagged PI3KC2β was co-transfected with either VN-tagged Ras17N, 17N/69N, or one the effector mutants in the background of Ras17N/69N. BiFC signal is pseudo-colored green. Effector mutations that disrupt Class I PI3K binding to Ras12V disrupt Class II PI3K binding to Ras17N/69N. CFP (red) was used as a transfection control. (C) Graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments. (*p = 0.02). (D) Western blot analysis demonstrates equal expression of all constructs. (E) Mutation of Thr392 to Asp or Lys379 to Ala in full-length PI3KC2β disrupts interaction with Ras17N. The ΔRBD mutant was also included as a negative control. Graph represents the average of three independent experiments (*p<0.05).</p

Biological activity of PI3KC2β versus Raf RBDs.

<p>(A) Expression of the Raf-RBD but not the PI3KC2β-RBD or GST alone inhibited EGF-stimulation of a Gal-Elk reporter assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045360#pone.0045360-Adams1" target="_blank">[23]</a>. GST-RBDs were expressed equally. GST alone migrated at a faster rate and was not visible in this image. Results represent the average relative activation ± S.E.M. from at least three independent experiments. (*p<0.05 compared to unstimulated GST, **p<0.01 compared to EGF stimulated GST). (B). PI3KC2β-RBD dose-dependently inhibits the effect of Ras17N on Src-mediated transformation. NIH/3T3 cells were transfected with 100 ng of SrcY527F expression construct in the presence or absence of Ras17N. Co-expression of the PI3KC2β-RBD reverses the inhibitory effect of Ras17N on Src transformation whereas the Raf-RBD does not. In contrast, expression of the Raf-RBD alone, but not the PI3KC2β-RBD, significantly inhibited Src-mediated transformation. The results represent the average relative focus forming activity ± S.E.M. from three independent experiments performed in triplicate. Asterisks denote samples that were significantly different from Src alone (*p<0.05).</p

Ras is necessary for ITSN1 activation of AKT.

<p>(A) YFP-ITSN1 overexpression stimulates AKT activation as measured by levels of phospho-AKT as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045360#pone.0045360-Das1" target="_blank">[22]</a>. Co-expression of Ras17N or Ras17N/69N inhibits this response. Results represent the average fold activation of AKT ± S.E.M. from at least three independent experiments. (B) Western blot analysis of AKT activation from a representative experiment. Top two panels represent Western blots of HA immunoprecipitates of cell lysates to assess AKT activation as described in the Materials and Methods section. The lower three panels indicate the level of expression of ITSN1, Ras, and actin (a loading control).</p

Ras, PI3KC2β, and ITSN1 co-localize on intracellular vesicles.

<p>(A) VN-Ras and VC-PI3KC2β (green) were co-transfected with CFP-ITSN1 (red) into COS cells. a. The PI3KC2β-Ras BiFC complex co-localizes with ITSN, represented by yellow in the overlay panel; b. The VN-Ras and VC-PI3KC2β YFP signal (green) does not bleed into the CFP channel; c. The CFP-ITSN1 signal does not bleed into the YFP channel (size bars  = 20 μm). Note: the differences in signal strength of the BiFC signal in (a) vs (b) are due to a lower power setting for the laser in (b) so that pixel intensities can be accurately quantified and are not saturated. In (a) a higher laser power was used to illustrate the punctate localization of the Ras-PI3KC2β complex throughout the cell. (B) Ras interaction with PI3KC2β is disrupted by deletion of the RBD (ΔRBD) but not by mutation of the Pro-rich, ITSN1 binding sites (PRD-PA). The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05, WT vs ΔRBD, PRD-PA vs ΔRBD). Western blot analysis demonstrates equal expression of all constructs (size bars  = 20 μm).</p

ITSN1 and Ras form a BiFC complex.

<p>(A) VC-tagged ITSN1 was co-transfected with one of the following VN-tagged Ras constructs: WT, 61L, 17N, or 17N/69N. ITSN1 formed a complex (green) with Ras17N >WT >17N69N >61L. CFP (red) was used as a transfection control. (B) The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05, **p<0.01). (C) A Western blot was performed to demonstrate equal expression of all constructs. (size bar  = 20 μm).</p

Model for ITSN1-Ras-PI3KC2β pathway.

<p>Growth factor stimulation of receptor tyrosine kinases leads to the recruitment of the Grb2-Sos complex resulting in dissociation of GDP from Ras. We propose that PI3KC2β competes for binding this transient nucleotide-free (nf) Ras trapping it in the nucleotide-free state and preventing GTP loading. In addition, binding of nucleotide-free Ras to PI3KC2β inhibits its lipid kinase activity. The PI3KC2β-Ras complex may then translocate to distal sites such as early endosomes (EE) where ITSN1 then binds to PI3KC2β leading to the release of nucleotide-free Ras and activation of the lipid kinase activity of PI3KC2β. In addition, once released from the ITSN1-PI3KC2β complex, Ras binds GTP resulting in Ras activation and recruitment of effectors, e.g., Raf, Class I PI3Ks, etc. Although the above model describes a potential role for nf-Ras in ITSN1 and PI3KC2β function, we propose that nf-Ras may be regulated by additional proteins besides these molecules. Our findings also raise the possibility that the nucleotide-free forms of other GTPases may play a similar role in cell signaling.</p

Ras forms a complex with PI3KC2β.

<p>(A) PI3KC2β preferentially interacts with Ras17N. VC-tagged PI3KC2β was co-transfected with one of the following VN-tagged Ras constructs: WT, 61L, 17N, or 17N/69N. BiFC signal (green) demonstrates that PI3KC2β interacted with Ras17N >17N/69N >WT >61L. CFP (red) was used as a transfection control. The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent (*p<0.05). Western blot analysis demonstrates equivalent expression of all constructs. (B) PI3KC2β does not interact with Ras12V. VC-tagged PI3KC2β was co-transfected with one of the following VN-tagged Ras constructs: WT,17N, or 12V. BiFC signal (green) demonstrates that PI3KC2β interacted with Ras17N >WT >12V. CFP (red) was used as a transfection control. The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05). Western blot analysis demonstrates expression of all constructs (size bar  = 20 μm).</p

Binding of PI3KC2β-RBD to Ras.

<p>(A) Nucleotide loaded Ras does not directly interact with the RBD of PI3KC2β. Ras-GDP or Ras-GTPγS were incubated with GST-Raf-RBD, GST-PI3KC2β-RBD or GST alone as a negative control. Bound proteins were then analyzed by Western blot with a Ras antibody. The RBD of Raf specifically bound Ras-GTPγS. Neither GST-PI3KC2β-RBD nor GST alone interacted with Ras-GDP or Ras-GTPγS. Top panel, Ras bound to GST proteins. Bottom panels, input amounts of proteins. (B) Nucleotide-free Ras was generated in vitro as described and then tested for binding to the various GST proteins as in (A). GST-PI3KC2β-RBD directly binds nucleotide-free Ras while little association was seen with the GST-Raf-RBD or GST alone. Panels are same as in A. (C) Repeat of (B) except nucleotide (1 mM) was present during the binding reaction. Panels are same as in (A). (D) Addition of nucleotide (1 mM) does not disrupt pre-bound PI3KC2β-RBD- nucleotide-free Ras. GST-PI3KC2β-RBD was first bound to nucleotide-free Ras. Following binding, the complex was incubated with 1 mM GDP or GTPγS at RT for 30 min and then washed with buffer. Bound proteins were then analyzed as in (A).</p

DataSheet1_Bioinformatic analysis of the LCN2–SLC22A17–MMP9 network in cancer: The role of DNA methylation in the modulation of tumor microenvironment.DOCX

Several features of cancer cells such as proliferation, invasion, metastatic spreading, and drug resistance are affected by their interaction with several tumor microenvironment (TME) components, including neutrophil gelatinase-associated lipocalin (NGAL), solute carrier family 22 member 17 (SLC22A17), and matrix metallopeptidase 9 (MMP9). These molecules play a key role in tumor growth, invasion, and iron-dependent metabolism of cancer cells. However, the precise epigenetic mechanisms underlying the gene regulation of Lipocalin 2 (LCN2), SLC22A17, and MMP9 in cancer still remain unclear. To this purpose, computational analysis was performed on TCGA and GTEx datasets to evaluate the expression and DNA methylation status of LCN2, SLC22A17, and MMP9 genes in different tumor types. Correlation analysis between gene/isoforms expression and DNA methylation levels of LCN2, SLC22A17, and MMP9 was performed to investigate the role of DNA methylation in the modulation of these genes. Protein network analysis was carried out using reverse phase protein arrays (RPPA) data to identify protein–protein interactions of the LCN2–SLC22A17–MMP9 network. Furthermore, survival analysis was performed according to gene expression and DNA methylation levels. Our results demonstrated that LCN2 and MMP9 were mainly upregulated in most tumor types, whereas SLC22A17 was largely downregulated, representing a specific hallmark signature for all gastrointestinal tumors. Notably, the expression of LCN2, SLC22A17, and MMP9 genes was negatively affected by promoter methylation. Conversely, intragenic hypermethylation was associated with the overexpression of SLC22A17 and MMP9 genes. Protein network analysis highlighted the role of the LCN2–SLC22A17–MMP9 network in TME by the interaction with fibronectin 1 and claudin 7, especially in rectal tumors. Moreover, the impact of expression and methylation status of LCN2, SLC22A17, and MMP9 on overall survival and progression free interval was tumor type–dependent. Overall, our analyses provide a detailed overview of the expression and methylation status of LCN2, SLC22A17, and MMP9 in all TCGA tumors, indicating that the LCN2–SLC22A17–MMP9 network was strictly regulated by DNA methylation within TME. Our findings pave the way for the identification of novel DNA methylation hotspots with diagnostic and prognostic values and suitable for epi-drug targeting.</p