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Activities of Dysregulated ALK2 Receptor Kinases Provide Insight Into the Protein Structural‐Functional Basis of Fibrodysplasia Ossificans Progressiva
Author(s) -
Groppe Jay Clemens,
TandangSilvas Mary Rose,
Pathi Anupama,
Wu Jingfeng,
Lu Guorong,
Le Viet Quoc,
Culbert Andria L,
Wharton Kristi A,
Shore Eileen M,
Kaplan Frederick Samuel
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.856.9
Subject(s) - kinase , microbiology and biotechnology , protein kinase domain , phosphorylation , mutant , smad , protein kinase a , biology , chemistry , wild type , genetics , gene
Although structurally similar to type II counterparts, type I or Activin‐like receptors (ALKs) are set apart by a metastable helix‐loop‐helix (HLH) element preceding the protein kinase domain that serves passive roles as an inhibitor‐to‐substrate binding switch according to a longstanding paradigm. A single recurrent mutation in the codon of the penultimate residue, directly adjacent the position of a constitutively activating substitution, causes milder ligand‐independent activation of ALK2 that leads to heterotopic bone deposition in patients presenting with FOP. Structure‐based homology modeling showed that the mutation (R206H) disrupts a conserved ion pair juxtaposed between an inhibitory protein (FKBP12) binding site and a loop conferring specificity for effector substrates (R‐Smads1/5/8). To determine the basis of the gain‐of‐function effect, wildtype, R206H mutant, Q207D (aspartate‐substituted) and HLH subdomain‐truncated (208ntrunc) forms were compared through in vitro kinase and protein‐protein interaction analyses that were complemented by signaling read‐out (p‐Smad) in primary mouse embryonic fibroblasts and Drosophila S2 cells. We observed that FKBP12 buffered, but did not strictly inhibit kinase activities in vitro, and had no detectable effect on basal signaling in wildtype fibroblasts. Contrary to paradigm, the HLH subdomain alone diminished the activity of wildtype kinase relative to mutant and variant forms. In addition, an absolute requirement for the HLH subdomain was observed for Smad phosphorylation despite significantly elevated kinase activity upon truncation. Hence rather than passively serving as obligate sites of binding and phosphorylation, the regulatory element plays active and direct roles in autoinhibition of kinase and recruitment of substrate, safeguarding through coupling the processes. Support or Funding Information This work was supported in part by grants from the National Institutes of Health (1R03 AR056838) and the UPenn Center for Research in FOP and Related Disorders to JCG. Hypothetical complex of the inhibitory protein FKBP12 ( blue ), the regulated type I BMP receptor kinase ALK2 ( gold ) and the constitutively active type II BMP receptor kinase BMPRII ( green )The FKBP12:ALK2 kinase complex, with the ATP‐competitive inhibitor dorsomorphin (compound C) bound, was experimentally determined (SGC Oxford; 3H9R). The BMPRII kinase crystal structure, with ADP ·Mg ++ bound (SGC Oxford; 3G2F), was docked to the ALK2 kinase through a server‐based computational routine.