(A) HPK1-interacting proteins identified using an antibody-based array and cell lysates from Panc-1/HPK1 stable cells treated with 2.0 μM MG132 to stabilize HPK1 protein. Immunodetection was performed using anti-FLAG antibody. (B) HPK1 interacts with RasGAP in Panc-1/HPK1 stable cells (C1) treated with 2.0 μM MG132. HPK1 kinase activity and autophosphorylation (p-HPK1^S171) are shown in the lower two panels. (C) GST pull-down assays show that RasGAP SH2-SH3-SH2 domains can effectively pull down HPK1 protein. No HPK1 was pulled down using RasGAP SH2 domain or GST alone. (D) RasGAP is phosphorylated at serine/threonine residue(s) by HPK1 but not M46. HPK1 or M46 was transfected into HEK293T cells. RasGAP was immunoprecipitated and probed with phospho–serine/threonine antibody. (E and F) In vitro RasGAP activity assays show that HPK1 enhances both basal and EGF-stimulated RasGAP activity as reflected by increased GDP/GTP ratio while M46 inhibits both basal and EGF-stimulated RasGAP activity. FLAG-HPK1 or FLAG-M46 was cotransfected with HA-Ras into HEK293T cells. Transfected cells were either untreated or treated with 100 ng/mL EGF for 10 minutes. The effect of HPK1 on RasGAP activity was determined by measurement of GTP- and GDP-bound Ras using thin-layer chromatography (E). (F) GTP- and GDP-bound Ras was quantified using ImageJ (NIH), and GDP/GTP ratios were plotted. Experiments were repeated 3 times. Data are presented as mean ± SEM. Statistical significance was determined by paired-samples t test using the cells transfected with Kras alone either untreated or treated with EGF as controls. When untreated, cotransfection of M46 with Kras significantly reduced the GDP/GTP ratio. Cotransfection of HPK1 with Kras increased the GDP/GTP ratio, but the P value was not significant. When treated with EGF, cotransfection of HPK1 with Kras significantly increased the GDP/GTP ratio, while cotransfection of M46 with Kras significantly reduced the GDP/GTP ratio. *P < 0.05; **P < 0.01.