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Crosstalk of cGMP dependent kinase I and BMP signaling
Eva Heining, Martina Weigt, Raphaela Schwappacher, Barak Marom,
Yoav Henis, Petra Knaus
This work is supported by the German-Israeli Foundation for Scientific Research and Development.
(GIF, grant number 932-244.13/2006)
The BMP type II receptor is expressed in two different splice variants, a long form (BRII-LF) that contains a long cytoplasmic tail after the kinase domain, and a short form (BRII-SF), which lacks this C-terminal extension. In a proteomics-based approach, new interacting partners of BRII-LF were identified, among them cGMP-dependent kinase I (cGKI). This cytoplasmic serine/threonine kinase also exists in two splice forms, which are encoded by the Prkg1 gene and differ in their first 100 amino acids. The protein consists of four putative domains: a cGMP binding domain, a peptide binding domain, a kinase domain, and an N-terminal domain involved in dimerization, autoinhibition and autophosphorylation. cGKI signals via the NO/cGMP/cGK pathway and is involved in regulation of cell growth, differentiation and apoptosis.
As cGKI was shown to bind to BRII, this interaction gives rise to novel mechanisms of regulating BMP signaling. Our group could demonstrate that cGMP-dependent kinase I (cGKI) modulates BMP receptors and Smads, providing a novel mechanism for enhancing BMP signaling (Schwappacher et al., 2009). In response to BMP2, cGKI dissociates from the receptor complex, associates with activated Smads, and undergoes nuclear translocation. In the nucleus, cGKI, Smad1, and the general transcription factor TFII-I bind to promoters of BMP target genes, such as Id1, to enhance their transcriptional activation.
The main purpose of the current project is to elucidate the crosstalk of cGKI and BMP signaling in. To achieve this goal, different biochemical and cell biological methods are used. Influences of cGKI on downstream events in the BMP pathway will be analyzed, using specific antibodies against intermediary signaling molecules in western blot assays or fluorescence labelled antibodies in immunofluorescence assays. Reportergene-based approaches allow verifications of effects on transcriptional activity. The phosphorylation status of the proteins is investigated in in vitro kinase assays.
COLLABORATIONS
Barak Marom, Prof. Dr. Yoav Henis
Department of Neurobiochemistry, George S. Wise Faculty of Life Science, Tel Aviv University
Activation of BMP-receptors;
characterization of receptor domains important for oligomerization and
signaltransduction
Petra Knaus
The bone morphogenetic proteins (BMP) play important roles in
embryogenesis and normal cell growth. The BMP receptors
belong to the family of serine/threonine kinase receptors, whose
activation has been investigated intensively for the TGF-ß
receptor subfamily. We have studied the interactions between the BMP
receptors, the composition of the active receptor complex
and the role of the ligand in its formation. We demonstrated that the
oligomerization pattern of the BMP receptors is different to
the TGF-ß receptors and is more flexible and susceptible to modulation
by ligand (Gilboa et al., 2000). Using several complementary approaches,
such as co-immunoprecipitation, binding and crosslinking of
radiolabelled BMP-2 as well as immunofluorescence co-patching of
epitope tagged receptors we showed in live cells pre-existing heteromeric
and homomeric oligomers in the absence of ligand.
BMP-2 binding significantly increased oligomerization (except for the
BR-II homo-oligomer). In contrast to previous
observations on TGF-ß receptors, which were found to be fully
homo-dimeric in the absence of ligand, the BMP-receptors show
a much more flexible oligomerization pattern. This novel feature in the
oligomerization mode of the BMP receptors allows
higher variety and flexibility in their responses to various ligands as
compared with the TGF-ß receptors.
Furthermore we could show that receptor oligomerization determines Smad-dependent and Smad-independent signalling. Preformed receptor complexes composed of BRIa and BRII induce the Smad pathway while ligand induced receptor complexes induce the MAPK pathway (Nohe et al., 2002).
Localization of BMP receptors in distinct plasma membrane domains and its impact on BMP signaling
Anke Hartung, Jan Börgermann, Petra Knaus
Collaboration: Yoav Henis,
Department of Neurobiochemistry,
George S. Wise Faculty of Life Science, Tel Aviv University
Endocytosis of growth factor receptors has an important regulatory influence on diverse
signal transduction pathways. It controls mainly the number and activation state of cell surface
receptors and is initiated at specific plasma membrane domains, e.g. clathrin-coated pits (CCPs),
caveolae and lipid rafts. Recently, it was shown that TGF-ß and EGF signaling pathways depend on
endocytic events and vesicles in different aspects.
BMP2 signals via two transmembrane serine/threonine kinase receptors BRI and BRII. Our group could
show that the canonical Smad pathway is initiated at preformed receptor complexes (PFC, composed of
BRI and BRII), which exist in an inactive state in the plasma membrane and get activated upon BMP2
binding. BMP2 binding to monomeric BRI causes dimerization of this receptor which subsequently leads
to the recruitment of BRII into the signaling complex; these receptor complexes are called
BMP2-induced signaling complexes (BISC). The signaling cascade which is initiated at BISCs involves
p38-MAPK as opposed to Smads resulting in the induction of Alkaline phosphatase (ALP)
(Nohe et al., 2002).
The aim of our recent studies was to elucidate how these different signaling pathways are initiated
from PFCs and BISCs and which role specific plasma membrane substructures as well as endocytic
processes play in this.
The first step was to explore BMP receptor localization with regard to detergent-resistent membrane
(DRM) regions (caveolae and lipid rafts) or clathrin-coated pits. Therefore, diverse microscopic and
biochemical techniques were applied, e.g. separation of DRMs by gradient ultracentrifugation,
immunoprecipitation, immunofluorescence or immuno-electronmicroscopy.
To investigate the influence of the specific localization of the BMP receptor complex on signaling
events, several cellular-based assays were established and employed. Early steps of the BMP signaling
cascade could be observed by measuring the phosphorylation state of Smad1/5, continuation of BMP
signaling was investigated by means of a specific reporter gene assay and an activity assay for ALP.
To address the different plasma membrane regions which could hypothetically influence BMP receptor
endocytosis and signaling, several inhibitors were applied. Cholesterol depletion using
Methyl-ß-cyclodextrin (MßCD), Lovastatin or Nystatin affects most likely lipid rafts and caveolae
because cholesterol is highly enriched in these regions. Chlorpromazine was used to inhibit
specifically CCP-mediated endocytosis. Moreover, caveolin-specific assays, e.g. overexpression of
caveolin-1 (cav-1) or application of cav-1 specific siRNA, influence caveolae formation and
endocytosis.
COLLABORATIONS
In collaboration with the group of Prof. Y. Henis (Keren Bitton-Worms, Maya Mouler Rechtman),
the endocytosis levels of BMP receptors by means of fluorescence microscopy and FRAP analyses were
investigated.
We demonstrate that both receptor types undergo constitutive endocytosis via CCP, but only BRII
undergoes also caveolar-like internalization. Using several complementary approaches we could show
that (i) BMP2-mediated Smad1/5 phosphorylation occurs at the plasma membrane in non-raft regions
(ii) continuation of Smad signaling resulting in transcriptional response requires endocytosis via
the clathrin-mediated route (iii) BMP signaling leading to ALP-induction initiates from receptors
that fractionate into cholesterol-enriched detergent-resistant membranes.
Furthermore we show that BRII interacts with Eps15R, a constitutive component of CCPs and with
cav-1, the marker protein of caveolae.
Taken together the localization of BMP receptors in distinct membrane domains is prerequisite to
different endocytosis routes with specific impact on Smad-dependent and Smad-independent signaling
cascades (Hartung et al., MCB, in press).
Summary of the effects of BMP receptor localization on BMP signaling.
BMP receptors are localized in CCPs and lipid rafts/caveolae and the signaling mode is influenced by
their localization and internalization as illustrated. Whereas Smad1/5 is phosphorylated while
present at the plasma membrane, Smad-dependent and Smad-independent continuation of BMP signaling
relies on different plasma membrane domains and endocytic events.
BMP type II receptor:
phosphorylation and associated proteins
Sylke Hassel, Raphaela Schwappacher, Martin Roth, Sabrina Scholz,
Simone Schmitt, Nadine Hemmrich, Walter Sebald and Petra Knaus
Collaboration: Ulf Hellman and Serhiy Souchelnytskyi,
Ludwig Institute for Cancer research, Uppsala, Sweden
In contrast to the TGF-ß receptors, BMPs have a high affinity to their type I receptors
(BRI-a and BRI-b) and a low affinity for their type II receptors. BMPs are disulfide-linked
homodimers. Heterodimeric variants with only one functional low-affinity epitope behave as
BMP-antagonists (Knaus P. and Sebald W., 2001).
The BMP type II receptor BRII exists in two different splice variants, a short form of 530 amino
acids, which is very similar to other type II receptors of the TGF beta superfamily (Liu F. et al
1995), and a long form of 1038 amino acids which contains a very long C-terminal extension (Kawabata
M. et al 1995, Rosenzweig B.L. et al 1995).
To further study signalling initiated by BRII we performed a proteomics based approach to identify
BRII interacting proteins which subsequently will be analysed for their physiological impact.
Another focus of our work is to elucidate the BRII phosphorylation which might regulate the
activation state of the receptor and serve as docking sites for other signal transducing molecules.
Methods
The basis for our work is molecular biology to generate mutants or different expression constructs. For examining the cellular responses we perform reporter gene assays as well as westernblots or differentiation assays. Recently we established the real time PCR method to examine the regulation of BMP responsive genes.
High separation capacity of 2D-GE in combination with sensitivity of mass spectrometry allows efficient identification of interacting proteins. We performed a screen for interacting proteins using GST pull-down with three constructs of BMPR-II; the whole intracellular part, the kinase domain, or the C-terminal part only, followed by two-dimensional gel electrophoresis and MALDI TOF MS. We identified more than 30 proteins forming a complex with BMPR-II constructs (collaboration with Prof. Dr. Ulf Hellman, Ludwig Institute for Cancer Research. Uppsala, Sweden). Currently we are working on their impact on signalling.
To identify the phosphorylation of BRII in vivo labelling and phospho peptid mapping was performed (collaboration with Dr. Serhiy Souchelnytskyi, Ludwig Institute for Cancer Research. Uppsala, Sweden). Furthermore in vitro kinase assays elucidate auto-phosphorylation events. Mutations of the identified phosphorylation sites and tests for their functionality are ongoing.
Differentiation assay: Alkaline Phosphatase staining in C2C12 cells,
non-stimulated and after treatment with 50nM BMP2 for 3 days.
BMP and Vitamin D signaling in differentiation of mesenchymal stem cells
Darja Obradovic, Jessica Becker, Jessica Kopf, Petra Knaus
BMPs belong to the TGFß superfamily, regulating a large variety of biological responses in many different tissues during embryonic development and postnatal life. Several members such as BMP2, BMP6, and BMP7 are especially potent in stimulating osteoblastic differentiation and bone formation (Hartung, 2006; Hassel, 2004).
Vitamin D3 (1,25-dihydroxy vitamin D3) is a steroid hormone with a major role in regulation of calcium and phosphate homeostasis. In the bone, vitamin D3 modulates the synthesis of ligands and receptors for both TGFß and BMP pathways and induces the expression of numerous markers along the osteoblastic lineage. Vitamin D signaling has already been shown to share and/or compete for several downstream components with the TGFß pathway, such as Smad3 and Smad7 co-activators (Junn, 1999).
In our lab, we aim to expand our focus to integrate vitamin D receptor signaling into well described membrane-initiated processes and examine combined effects of the two osteogenic inducers (vitamin D3 and BMP analogues) in advancing osteoblastic differentiation. For this, we use human mesenchymal stem cells (MSC) as a model. Ongoing experiments focus on the effects of BMP2, vitamin D3 (natural and synthetic), and the combination of both ligands on undifferentiated and differentiated MSCs.
In parallel, the impact of vitamin D3 (1,25-dihydroxy vitamin D3 and various synthetic analogues) is investigated in terms of changes in BMP signaling, which reflects both genomic and non-genomic interplay between those two osteogenic inducers.
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