Extended Data Fig. 4: ZDHHC19 mediates STAT3 palmitoylaiton and Ras proteins are not involved in ZDHHC19-mediated STAT3 signalling.
From: Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation
a, b, HEK293A cells were co-transfected with construct encoding Flag–STAT3 and HA-tagged ZDHHC proteins. After 48 h, cells were labelled for 4 h with 50 μM palmitoylation probe (alk-C16). Cell lysates were reacted with biotin azide and subjected to streptavidin bead pulldown. STAT3 palmitoylation was shown by western blot. c, HEK293A cells were transfected with Flag–STAT3 alone or in combination with HA–ZDHHC19, HA–ZDHHC5 or HA–ZDHHC 18, and labelled with 50 μM probe (alk-C16) for 4 h, followed by analysis of STAT3 palmitoylation. d, HEK293A cells were transfected with Flag–STAT3 alone or in combination with HA–ZDHHC19, HA–ZDHHC5 or HA–ZDHHC18. Whole-cell lysates were analysed by anti-Flag immunoprecipitation, followed by immunoblotting using the indicated antibodies. e, HEK293A cells were transfected as in d. Total cell extracts were analysed by western blot after immunoprecipitation with anti-HA antibody. f, HEK293A cells were transfected with Flag-tagged wild-type STAT3 or Flag–STAT3(K685S), with or without ZDHHC19. Palmitoylation of STAT3 was analysed as in c. g, h, HEK293A cells were transfected with HA-tagged wild-type ZDHHC19 or the inactive mutant ZDHHC19(C142S), and labelled with 50 μM alk-C16 or C16–BYA. Cell lysates were subjected to streptavidin bead pulldown. STAT3 palmitoylation was showed by western blot. i, HEK293A cells were transfected with STAT3–luciferase reporter and Renilla control, co-transfected with wild-type ZDHHC19 or ZDHHC19(C142S) as shown, and treated with IL-6 (20 ng ml−1) and/or palmitic acid (100 μM) for 8 h. Luciferase activity was measured, and relative fold induction was plotted from triplicate experiments. j, HEK293A cells were co-transfected with ZDHHC19-shRNA and wild-type ZDHHC19 or ZDHHC19(C142S), and the expression of the indicated genes was quantified with qRT–PCR. k–m, KNS62 control cells and ZDHHC19-knockout stable cells were treated with IL-6 (20 ng ml−1) and/or palmitic acid (100 μM) for 8 h. The mRNA levels of STAT3 target genes (BCL2, BCL2L1 and MMP9) were analysed by qRT–PCR. n, Schematic of wild-type GRB2 and SH3-domain truncation mutants, and alignment of the SH3 binding motif sequences of ZDHHC19. o, HEK293A cells were co-transfected with MYC-tagged wild-type ZDHHC19 and GFP-tagged wild-type GRB2, or GRB2 with an N-terminal SH3 deletion (ΔN-SH3) or C-terminal SH3 deletion (ΔC-SH3). Whole-cell lysates were subjected to immunoprecipitation using anti-MYC antibody, followed by western blotting using the indicated antibodies. p, HEK293A cells were co-transfected with MYC-tagged wild-type ZDHHC19 or ZDHHC19(P18A) mutant, Flag-tagged STAT3 and GFP-tagged GRB2, followed by co-immunoprecipitation assay. q, HEK293A cells were treated with IL-6 (20 ng ml−1) and analysed by anti-STAT3 co-immunoprecipitation assay, followed by western blotting using the indicated antibodies. r, s, HA-tagged ZDHHC19 was co-transfected with HA-tagged HRAS (r) or NRAS (s) into HEK293A cells. After 24 h, cells were incubated with fresh medium containing 10% dialysed FBS for 2 h, and subsequently labelled with 50 μM probe (alk-C16) for 4 h. Cell lysates were reacted with biotin azide and subjected to SDS–PAGE. Streptavidin blot was used to detect HRAS (r) or NRAS (s) palmitoylation, as described for the detection of STAT3 palmitoylation in Methods section. Comparable protein loading was confirmed by anti-HA and β-actin western blotting. t, u, Treatment with the depalmitoylase inhibitor palmostatin B (10 μM) or overexpression of the depalmitoylase ABHD17A has no effect on the expression of STAT3 target genes (BCL2, BCL2L1 and MMP9) in HEK293A cells that express oncogenic HRAS(G12V) (t) or NRAS(G12V) (u). v, Treatment with different concentrations of palmostatin B (1 μM, 5 μM and 10 μM) has no effect on the expression of STAT3 target genes (BCL2, BCL2L1 and MMP9) in melanoma SK-MEL-2 cells that contain the NRASQ61R mutation. In i–m, t–v, the data are presented as mean ± s.e.m. n = 3 biologically independent samples. P values were determined by two-tailed Student’s t-test. In a–h, o–s, the experiments were independently repeated at least three times with similar results. For gel source data, see Supplementary Fig. 1.
