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. 2020 Jan 31;23(2):100871. doi: 10.1016/j.isci.2020.100871

Figure 6.

Figure 6

RET-MEN2A and -MEN2B Cancer Mutations and their Regulation by PTPRA

(A) RET-linked human multiple endocrine neoplasia (MEN) type 2 carcinomas, affecting the thyroid and adrenal glands. The corresponding MEN2 tumorigenic mutations (vertical red bars), MEN2A (C634W) and MEN2B (M918T), are in the RET CRD and TK domains, respectively. CLD1-4, cadherin-like domains 1–4; CRD, cysteine-rich domain; TM, transmembrane region; JM, juxtamembrane region; and TK, tyrosine kinase domain.

(B) PTPRA co-immunoprecipitates with both RET-MEN2A and -MEN2B mutants. V5-tagged RET-MEN2A or -MEN2B and either StrepIII-HA-tagged wild-type (WT) or phosphatase domain-deleted (DD: ΔD1 and ΔD2) mutants of PTPRA were expressed in HEK293-MSR cells. The immunoprecipitated (IP) proteins, as well as cell lysates, were immunoblotted with anti-V5 and anti-HA antibodies to detect RET-MEN2s and PTPRA proteins, respectively. Tubulin is used as a loading control (* unspecific band).

(C) In vivo tyrosine phosphorylation of RET and MEN2A, but not of MEN2B, successively decreases with the increasing amounts of PTPRA (0, 2.5, and 5 μg) construct. The RET and MEN2 (A and B) proteins were precipitated from cell lysates with anti-HA beads and immunoblotted with general anti-phosphotyrosine (pTyr) antibody. Equal gel loading is verified by re-probing with anti-HA antibody, and relative intensity of pTyr level is analyzed by ImageJ software. PTPRA expression in cell lysate is determined by the anti-V5 antibody. Tubulin is used as a loading control.

(D) PTPRA efficiently inhibits the wild-type RET and MEN2A, whereas the MEN2B is less sensitive to the inhibition in the Ras-MAPK reporter assay, shown in the absence (blue bars) and presence (orange bars) of GDNF-GFRα1 ligands (100–500 ng/mL; 24 h). The bar graph is representative of two independent experiments, where all bars indicate average ratios (n = 5 replicates) of Firefly to Renilla luciferase counts (relative luciferase unit, RLU) and the error bars designate one standard deviation. The statistical significance is obtained by a two-tailed Student's t test (***p < 0.0001 and **p < 0.001). See also Table S3.

(E) Similarly to the wild-type RET, the ΔD2 mutant inhibits MEN2A but not MEN2B in the absence (blue bars) and presence (orange bars) of GDNF-GFRα1 ligands. Interestingly, the ΔD1 mutant slightly increases the MEN2B activity in our reporter assay. The bar graph is representative of two independent experiments, where all bars indicate average ratios (n = 5 replicates) of Firefly to Renilla luciferase counts (relative luciferase unit, RLU) and the error bars designate one standard deviation. The statistical significance is obtained by a two-tailed Student's t test (***p < 0.0001 and **p < 0.001).