Fig 1: KSR1 regulates YAP subcellular localization.A WT, KSR1+/+ and KSR1−/− MEFs were lysed and fractionated. Equal amounts of cytoplasmic (Cyt) and nuclear (Nuc) protein were resolved by Western blotting and probed with the indicated antibodies. HSP90 and HDAC were controls for cytoplasmic and nuclear fractions, respectively. B YAP bands were quantified and corrected for HSP90 (cytoplasmic fraction) or HDAC (nuclear fraction). Data are expressed as means ± S.E. (N = 3), with WT MEFs set to 1. *p < 0.05, ****p < 0.0001, Student’s t test. C WT and KSR1–/– MEFs were fixed and stained with anti-YAP monoclonal antibody (green). Actin was visualized using Alexa Fluor® 568 Phalloidin (red) and DNA was stained with Hoechst 33342 (blue). Representative confocal images are shown from at least 100 cells imaged from three separate biological samples. Insets show higher magnification of the selected areas (right panels). Scale bar, 10 μm. White arrows show areas of actin protrusions and cell aggregates. D Python (v.3.10) was used to identify and segment the nuclear regions based on Hoechst staining. The intensity of YAP staining in the nucleus in each cell was quantified. Data are means ± S.E. (N = 3, 150 cells). ****p < 0.0001, Mann–Whitney test.
Fig 2: KSR1 modulates YAP function via RhoA and the actin network.A WT, KSR1+/+ and KSR1−/−MEFs were fixed and stained with anti-phosphoPaxillinTyr118 (pPaxillin) monoclonal antibody (green) to visualize focal adhesions. Actin was visualized using Alexa Fluor® 568 Phalloidin (red), and DNA was stained with Hoechst (blue). Representative confocal images are shown from at least 100 cells imaged from three separate biological samples. Insets show higher magnification of the boxed area. Scale bar, 10 μm. B Actin stress fibers were segmented and measured as described in the methods. ****p < 0.0001, Kruskal-Wallis test. C–E Nuclear regions were segmented based on Hoechst staining and geometric parameters of nuclear segments, including area, eccentricity (ratio of minor axis length to major axis length) and solidity (ratio of the region area to area of the convex hull), were measured. The extent of nuclear displacement is defined as the Euclidean distance between the nuclear centroid and the cytoplasmic centroid. Data are expressed as medians ± 95% CI (B) and means ± S.E. C–E from at least 100 cells measured in three separate biological samples. **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test. ns, not significant. F WT (left panel) and KSR1−/− (right panel) MEFs starved of serum for 16 h were incubated with (+) or without (–) FBS for 5 min. RhoA-GTP (active RhoA) was quantified using the G-LISA assay. Data represent means ± S.E., ****p < 0.0001, Student’s t test (N ≥ 4 biological samples), with values in starved cells set to 1. G WT and KSR1–/– MEFs were starved of serum for 6 h, then incubated with either vehicle (ddH2O) or the RhoA activator CN03 for 4 h. Cells were fixed and processed as described for (A). H Serum-starved KSR1+/+ MEFs were incubated with the RhoA inhibitor CT04 or vehicle (ddH2O) for 4 h. Cells were processed as described for (A). All confocal images are representative of at least 100 cells imaged from three separate biological samples. Scale bar, 10 μm.
Fig 3: Model of KSR1 in Hippo signaling.A KSR1 constitutively binds MST1, YAP and LATS1, components of the Hippo pathway. (i) KSR1 binds directly to YAP, potentially protecting YAP from phosphorylation by kinases of the Hippo cascade, thereby enabling YAP to translocate to the nucleus. (ii) KSR1 binds directly to MST1 and interferes with MST1 dimerization and consequent autophosphorylation/activation. (iii) KSR1 enhances YAP mRNA expression. (iv) KSR1 enhances activation of RhoA and RhoA/Rho-associated protein kinase (ROCK). This modulates actin organization, suppresses LATS1 activation and promotes YAP nuclear localization. (v) Depletion of KSR1 increases the amounts of phosphorylated (active) MST1 and LATS1, which increases YAP phosphorylation and reduces YAP function by blocking its translocation to the nucleus. B In response to EGF, KSR1 scaffolds the MAPK pathway by assembling a functional complex of Raf, MEK and ERK. EGF attenuates the association of KSR1 with MST1, LATS1 and YAP, thus releasing the inhibitory impact of KSR1 on Hippo signaling. The KSR1 is then free to scaffold components of the MAPK cascade. Green arrows and red lines represent activation and inhibition, respectively. EGF epidermal growth factor, EGFR epidermal growth factor receptor, ROCK Rho-associated protein kinase, PM plasma membrane, TF transcription factor. The figure was generated in BioRender.
Supplier Page from Abcam for Recombinant Human KSR1 protein (Tagged)