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. 2019 Jun 21;218(8):2470–2480. doi: 10.1083/jcb.201903068

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© 2019 Centonze et al.

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Figure 4. — LTK interacts with and phosphorylates Sec12. (A) Volcano plot of the interactome of HA-tagged LTK revealed by immunoprecipitation MS from HEK293 cells. The red labeled candidate interacting proteins are related to receptor tyrosine kinase signaling or ER-associated processes. The table indicates the top-scoring biological processes enriched among the LTK interaction partners. (B) Schematic representation of key components of the ER folding, quality control, and export machinery with red-highlighted interaction partners. Red box highlights ER export. (C) Flag-tagged LTK was immunoprecipitated followed by immunoblotting against Sec12, which was identified in the interactome and against RTN4, a transmembrane protein that we did not find in the LTK interactome. (D) HeLa cells were transfected with vectors encoding flag-tagged Sec12 or its mutants together with an empty vector or with YFP1-tagged LTK (LTK-Y1). In the last lane are lysates from cells pretreated with 1 µM crizotinib for 30 min. (E) Sequence logo to demonstrate the conservation of amino acids in Sec12 in mammals or in metazoan excluding vertebrates. (F) Purified GST-tagged cytosolic domain of Sec12 or GST were incubated a flag-LTK immunoprecipitate from HEK293 cells (LTK immunoprecipitation) in the presence or absence of ATP or crizotinib.