Fig. 7: ABL1/2 phosphorylate MAP3K1 and 14-3-3-ε. | Nature Communications

Fig. 7: ABL1/2 phosphorylate MAP3K1 and 14-3-3-ε.

From: Combating acquired resistance to MAPK inhibitors in melanoma by targeting Abl1/2-mediated reactivation of MEK/ERK/MYC signaling

Fig. 7

a 293T cells were transfected with His-MAP3K1 and/or constitutively active forms of ABL1 or ABL2 (PP), His-MAP3K1 pelleted with nickel agarose, complexes blotted with phospho-tyrosine (pTyr) antibody, and blot stripped and reprobed with control antibodies. Blots shown are representative of n = 3 independent experiments. b Endogenous MAP3K1 or 14-3-3 (pan) were immunoprecipitated from M14-BMR cells, and IPs blotted with the indicated antibodies. Blots shown are representative of n = 2 independent experiments. c Recombinant forms of ABL1 or ABL2 were incubated with full-length, recombinant MAP3K1 in a “cold” in vitro kinase assay, and reactions blotted with phospho-tyrosine (pTyr) antibody, stripped and reprobed with antibody to MAP3K1. Blots shown are representative of n = 2 independent experiments. d Recombinant proteins were incubated in a kinase assay as in c using either His-MAP3K1, GST-14-3-3-ε, or the combination as substrates. The phospho-tyrosine blot (top) was stripped and reprobed with antibody directed at the MAP3K1 C-terminus (middle) or GST (bottom; to visualize 14-3-3). Blots shown are representative of n = 2 independent experiments. e Scheme showing mechanism by which ABL1/2 drive ERK reactivation and resistance (red arrows).

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