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Recurrent somatic mutations underlie corticotropin-independent Cushing’s syndrome

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Science  23 May 2014:
Vol. 344, Issue 6186, pp. 917-920
DOI: 10.1126/science.1252328

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  1. Fig. 1 Recurrent mutations in PRKACA and GNAS.

    (A) Mutations in PRKACA (top) and GNAS (bottom) identified in 65 patients with corticotropin-independent Cushing’s syndrome (arrowheads). Confirmed somatic mutations are indicated in red. (B) Mutually exclusive distribution of PRKACA and GNAS mutations.

  2. Fig. 2 Relationship between mutation status and clinical features.

    (A and B) Serum cortisol level of 1-mg dexamethasone (A) and the diameter of adrenocortical adenoma (B) according to the mutation status of PRKACA/GNAS genes. (C) Clinical and subclinical Cushing’s syndrome as attributed to mutated PRKACA, GNAS, or other, currently undetected, cause.

  3. Fig. 3 Effect of L206R mutation on three-dimensional structures of PKA.

    (A) Three-dimensional structure of the PKA complex, composed of C (PRKACA) (pink) and R (PRKAR1A) (cyan) subunits, is depicted using the University of California–San Francisco Chimera program, based on the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB ID: 2QCS). L206 is shown in red. (B and C) A predicted effect of the L206R mutation within the P+1 loop on the interaction with the R subunit (C) in comparison with wild-type PRKACA (B).

  4. Fig. 4 Functional characterization of PRKACA mutant.

    (A) Immunoblot analysis of antibody to FLAG immunoprecipitates of the PKA reconstructed in vitro with large-scale–purified proteins, in which PRKACA and PRKAR1A subunits are tagged with hemagglutinin (HA) and FLAG, respectively. (B) In vitro PKA activities of wild-type and mutant PRKACA in the presence or absence of PRKAR1A and cAMP with purified proteins. (C) Immunoblot analysis of the total cell lysates (input) and antibody to HA immunoprecipitates from HEK293T cells stably transduced with either mock, wild-type, or mutant PRKACA tagged with HA with indicated antibodies. (D) PKA activities in HEK293T cells stably transduced with either mock, wild-type, or mutant PRKACA in the presence or absence of forskolin stimulation. (E) Relative expression of pCREB to total CREB in HEK293T cells stably transduced with either mock, wild-type, or mutant PRKACA as determined by densitometory of the immunoblots. (F) The effect of inhibition of PKA (H89 and KT5720) and cAMP (Rp-cAMPS) in HEK293T cells transduced with mock or mutant PRKACA. (G) PRKACA expression standardized for mRNA expression in PRKACA-mutated and unmutated adenomas and matched normal adrenocortical tissues. (H) Western blot analysis of indicated fractions of cell lysates from wild-type and mutant PRKACA-transduced HEK293T cells using indicated antibodies. Standard errors and significant differences are indicated [(C) to (G)].

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