Fig 1: Proposed model of MAP4K4 and STRN3 regulation of cell migration and proliferation in MB.MAP4K4 is activated downstream of RTK signaling and exerts both pro-invasive and anti-proliferative activities in MB cells. Invasion regulation: STRN3 cooperates with MAP4K4 to control cell migration, invasion, and actin cytoskeleton remodeling, independently from PP2A activity. This cooperation of MAP4K4 and STRN3 is necessary for the FGFR-induced activation of motility-associated effectors nPKCs and VASP. Proliferation regulation: MAP4K4 causes LATS1/2 phosphorylation, which promotes the activation of the Hippo pathway. Hippo pathway activation results in the phosphorylation and degradation of the transcriptional regulator YAP. The consequent repression of YAP transcriptional activity leads to a proliferation arrest. STRN3 promotes PP2A-mediated inactivation of MAP4K4 function toward Hippo activation, resulting in an increase of YAP transcriptional activity and cell proliferation.
Fig 2: MAP4K4 and STRN3 mediate VASPS157 phosphorylation.a Heat map of increased (red) and decreased (blue) phosphorylation of Ser/Thr (STK, left) or Tyr (PTK, right) consensus peptides associated with lysates from siRNA transfected DAOY cells treated ± 100 ng/ml bFGF for 15 min. Log2 of the fold change between siTarget and siCTRL (n = 3). b Venn diagram displaying the number of Ser/Thr and Tyr peptides with significantly changed phosphorylation (p < 0.1) in each siTarget versus siCTRL with bFGF stimulation (n = 3). c Volcano plots representing the changes in phosphorylation of STK (circles) and PTK (diamonds) peptides in siMAP4K4 (upper) or siSTRN3 (lower) versus siCTRL with bFGF stimulation. Blue: peptides with significantly decreased (fold change < 0.6), red: peptides with significantly increased phosphorylation (fold change > 1.67). The p-values were calculated versus siCTRL by ANOVA and post hoc Dunnett’s test in the BioNavigator software. n = 3. d Single confocal section of VASP localization in DAOY cells seeded on collagen-coated plates ± 100 ng/ml bFGF for 15 min. 3× magnifications of boxed areas are shown. Red: VASP, Green: Lifeact-EGFP. Blue: DNA Hoechst. Scale bar: 20 µm. e IB and quantification of VASPS157 phosphorylation in DAOY cells 72 h after siRNA transfection ± 100 ng/ml bFGF for 15 min (n = 4, means ± SEM). f IB and quantification of VASPS157 phosphorylation in DAOY cells expressing full-length or truncated (KD-ID1-ID2) MAP4K4-FLAG ± 100 ng/ml bFGF for 15 min (n = 3, means ± SEM). g In vitro VASP S157 phosphorylation of FLAG-immunoprecipitated DAOY cells expressing FLAG-tagged MAP4K4 and transfected for 48 h with the indicated siRNA ± 100 ng/ml bFGF for 15 min. The in vitro kinase reaction was conducted incubating the IP fraction with a recombinant VASP protein. Parental WT DAOY cells were used as a negative control for the IP. h Quantification and representative images of SIA of siCTRL or siVASP transfected DAOY cells ± 100 ng/ml bFGF (n = 4, means ± SEM). Scale bar: 300 µm. Statistical analysis in e, f was performed by unpaired Student’s t-test, in h by one-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Fig. S9. Source data for quantifications shown in (e), (f), and (h) are available in Supplementary Data 10.
Fig 3: (A) Venn diagram showing overlap of Ag for autoimmune diseases of the nervous system and hub genes in blue and saddle brown modules. (B–D) Secondary structure prediction, hydrophilicity, antigenic index, and surface probability with aligned fragments plots of VASP (B), ANX11 (C), and CBP (D).
Fig 4: (A) ELISA of anti-VASP autoantibodies with samples classified as SLE/HC or NPSLE/other SLE/HC. The vertical axis represents the normalized OD values. (B) Western blot showing the expression of anti-VASP autoantibodies. The green arrow indicates the position of positive control (complex of VASP and primary Ab), and the red arrow indicates the immunocomplex of VASP and its autoantibody from serum. (C) Protein-protein interaction network and functional enrichment of VASP and its adjacent proteins in blue/saddle brown modules. (D) The Correlation Matrix between anti-VASP Antibodies and psychoneurological symptoms.
Supplier Page from Abcam for Recombinant Human VASP protein (His tag N-Terminus)