Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor
Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR‐II) which has a serine/threonine...
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| Published in | Journal of cellular physiology Vol. 206; no. 2; pp. 457 - 467 |
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| Main Authors | , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Hoboken
Wiley Subscription Services, Inc., A Wiley Company
01.02.2006
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| Subjects | |
| Online Access | Get full text |
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| Summary: | Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR‐II) which has a serine/threonine kinase domain, and stem cell factor receptor (c‐kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2‐dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2‐dependent differentiation of C2C12 cells. We found that BMPR‐II was phosphorylated at Ser757 upon co‐expression with and activation of c‐kit. BMPR‐II phosphorylation required intact kinase activity of BMPR‐II. Abrogation of the c‐kit/SCF‐dependent phosphorylation of BMPR‐II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c‐kit and BMPR‐II leads to phosphorylation of BMPR‐II at Ser757, which modulates BMPR‐II‐dependent signaling. J. Cell. Physiol. 206: 457–467, 2006. © 2005 Wiley‐Liss, Inc. |
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| Bibliography: | GST constructs which were used for a GST pull-down experiment.GST constructs of BMPR-II and GST alone, which were used for pull-down of c-kit, are shown. Migration positions of BMPR-II constructs and GST alone are indicated by *, and by arrows. This is Figure 1A from Hasselet al . (2004), as the same constructs were used by Hasselet al . (2004).Kinase deficient mutant of c-kit formed a complex with HA-BMPR-II.Wild-type HA-BMPR-II (BR-II) and wild-type (c-kit) or kinase deficient (c-kit KN) constructs of c-kit were transfected in 293T cells, as indicated. Receptors were immunoprecipitated with anti-c-kit or anti-HA antibodies, subjected to SDS-PAGE, and HA-BMPR-II was detected by immunoblotting with anti-HA antibodies (upper panel). HA-BMPR-II co-precipitated with wild-type c-kit (c-kit) and kinase-deficient mutant of c-kit (c-kit KN), and HA-BMPR-II precipitated with anti-HA, as a control, are indicated with an arrow (upper panel). Expression of c-kit and HA-BMPR-II constructs was monitored by immunoblotting of whole cell lysates with anti-HA (middle panel) or anti-c-kit (lower panel) antibodies. Migration positions of HA-BMPR-II and c-kit are shown by arrows. Note lower expression level of kinase deficient c-kit protein, which correlates with weaker signal for HA-BMPR-II co-precipitated with c-kit KN. Representative experiment out of 2 performed, is shown.Various anti-BMPR-II antibodies have different efficiency for co-precipitation of endogenous c-kit with BMPR-II.Whole C2C12 cell lysates were subjected to immunoprecipitation with various anti-BMPR-II antibodies, as indicated. c-Kit was detected by immunoblotting with anti-c-kit antibodies. We observed that the "1805" antibodies were most efficient in co-precipitation of c-kit. This antibody was used in further studies which are reported in this manuscript. To monitor the efficiency of c-kit detection, we loaded onto the gel whole cell extract from cells transfected with c-kit and HA-BMPR-II (c-kit/BRII transfected cells; right lane), as indicated. Migration position of c-kit is shown by an arrow.SCF does not affect BMP2-dependent inhibition of C2C12 cells proliferation.C2C12 cells were treated with BMP2 and SCF, as indicated. We observed an inhibitory effect of BMP2 on proliferation of C2C12 cells, and no synergistic effect of SCF was observed. 1x103 C2C12 cells were plated on a 96-well plate. The next day, they were starved for 5h (DMEM, 0.5% FCS) and subsequently stimulated with 500 ng/ml BMP2, 100 ng/ml SCF or both ligands for 24h. Proliferation was measured using the CellTiter 96® AQueous one solution (Promega), according to manufacturer's instructions. Representative experiment out of 5 performed, is shown.Phosphorylation of c-kit at tyrosine residues is not affected by BMPR-II.Phosphorylation of c-kit on tyrosine residues is not affected upon co-expression of BMPR-II wild type (BRII) or kinase deficient receptor (BRII-KR), and treatment with BMP2. BMPR-II and c-kit receptors were transfected in COS7 cells, as indicated. Cells were treated with BMP2 (500 ng/ml) and SCF (100 ng/ml), as indicated. c-Kit receptor was immunoprecipitated from cell lysates, and immunoblotted with pY99 anti-phosphotyrosine antibody (upper panel). Expression of c-kit was monitored by immunoblotting with anti-c-kit antibody (lower panel). Migration position of c-kit is shown by arrows.BMPR-II phosphopeptide which appears upon co-expression of c-kit and treatment of cells with both BMP2 and SCF, contains a phosphorylated residue at the position 4.Radiochemical sequencing of the c-kit-dependent phosphopeptide was performed as described earlier (Yakymovychet al. , 2001). The image shows radioactivity eluted in the fourth cycle of the radiochemical sequencing, as indicated by an arrow.Phosphorylation pattern of c-kit is not affected by co-expression of BMPR-II or by treatment of cells with BMP2.Transfection of 293T cells, metabolic labelling with [32P]orthophosphate, treatment with BMP2 and SCF, and two-dimensional phosphopeptide mapping of phosphorylated c-kit were performed, as described in Figure 5 for BMPR-II. Analysis of phosphopeptide maps of c-kit showed that SCF induced appearance of additional phosphopeptides, but BMP2 did not affect the phosphorylation pattern. Royal Swedish Academy of Sciences Swedish Research Council istex:8026964FDC22550C9BCA9F0C0F5669DB539E72CE INSERM/VR EU programs "T-Anigiovasc" and RTN "EpiPlastCarcinoma" Hiroshima University ArticleID:JCP20480 This article includes Supplementary Material available from the authors upon request or via the Internet at http://www.interscience.wiley.com/jpages/0021-9541/suppmat. Merck KgaA Swedish Cancer Society ark:/67375/WNG-78CPHZNF-P Boehringer Ingelheim Fonds This article includes Supplementary Material available from the authors upon request or via the Internet at http://www.interscience.wiley.com/jpages/0021‐9541/suppmat . ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0021-9541 1097-4652 1097-4652 |
| DOI: | 10.1002/jcp.20480 |