Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry

• Bone morphogenetic proteins (BMP) are polypeptide growth factors that regulate cell differentiation and proliferation. BMPs bind to type I and type II serine/threonine kinase receptors to initiate intracellular signalling. BMPR-II is the type II receptor, its mutations lead to hereditary pulmonary hypertension, and knockout of Bmpr-II results in early embryonic lethality. To identify novel interacting proteins and explore signalling pathways that can be initiated by BMPR-II, we performed glutathione-S-transferase (GST) pull-down assays with BMPR-II protein constructs fused to GST and extracts of mouse myoblast C2C12 cells. We generated three constructs which contain different parts of the cytoplasmic region of BMPR-II: full-length cytoplasmic part of BMPR-II, only the kinase domain, or only the C-terminal tail of BMPR-II. Proteins which formed complexes with these BMPR-II constructs were analyzed by two-dimensional gel electrophoresis (2-D GE), and specifically interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). We identified 33 interacting proteins; 11 proteins interacted with the C-terminal tail of BMPR-II, 4 with full-length BMPR-II, and 18 with a short form of the receptor with a deleted tail. Fourteen proteins have assigned functions in various signalling processes, suggesting links of BMP signalling to regulation of MAP kinase pathway, apoptosis, transcription, PKCss, and PKA. Five of the identified proteins are components of the cytoskeleton, and four are enzymes involved in metabolism, e.g., processing of estrogens or lipids. We confirmed interaction of PKC beta and CtBP with BMPR-II using immunodetection. We showed that the C-terminal tail of BMPR-II provides binding sites for a number of regulatory proteins that may initiate Smad-independent signalling.

Nyckelord

Amino Acid Sequence

Animals

COS Cells

Cell Extracts/chemistry

Cell Line

Cercopithecus aethiops

Chemiluminescent Measurements

Cysteine/metabolism

DNA-Binding Proteins/immunology

Electrophoresis; Gel; Two-Dimensional

Glutathione Transferase/metabolism

Methionine/metabolism

Mice

Myoblasts/metabolism

Phosphoproteins/immunology

Precipitin Tests

Protein Kinase C/immunology/isolation & purification

Protein Structure; Tertiary

Protein-Serine-Threonine Kinases/*analysis/chemistry/*metabolism

Proteins/*analysis/metabolism

Recombinant Fusion Proteins/metabolism

Research Support; Non-U.S. Gov't

Silver Staining

Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization

Spectrum Analysis; Mass

Sulfur Radioisotopes/metabolism

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