Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.

Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser/Thr kinase-mediated TGF-beta signaling. The crystal structure of a phosphorylated Smad2, at 1.8 A resolution, reveals the formation of a homotrimer mediated by the C-terminal phosphoserine (pSer) residues. The pSer binding surface on the MH2 domain, frequently targeted for inactivation in cancers, is highly conserved among the Co- and R-Smads. This finding, together with mutagenesis data, pinpoints a functional interface between Smad2 and Smad4. In addition, the pSer binding surface on the MH2 domain coincides with the surface on R-Smads that is required for docking interactions with the serine-phosphorylated receptor kinases. These observations define a bifunctional role for the MH2 domain as a pSer-X-pSer binding module in receptor Ser/Thr kinase signaling pathways.

[1]  P. Hoodless,et al.  MADR2 Is a Substrate of the TGFβ Receptor and Its Phosphorylation Is Required for Nuclear Accumulation and Signaling , 1996, Cell.

[2]  J. Massagué,et al.  Determinants of specificity in TGF-beta signal transduction. , 1998, Genes & development.

[3]  Jeffrey L. Wrana,et al.  Mechanism of activation of the TGF-β receptor , 1994, Nature.

[4]  John Kuriyan,et al.  Crystal structure of the Src family tyrosine kinase Hck , 1997, Nature.

[5]  R. Fairman,et al.  Formation of a Stable Heterodimer between Smad2 and Smad4* , 2001, The Journal of Biological Chemistry.

[6]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[7]  J. Darnell,et al.  Crystal Structure of a Tyrosine Phosphorylated STAT-1 Dimer Bound to DNA , 1998, Cell.

[8]  J. Massagué,et al.  Structural basis of Smad2 recognition by the Smad anchor for receptor activation. , 2000, Science.

[9]  Jeffrey L. Wrana,et al.  TβRI Phosphorylation of Smad2 on Ser465 and Ser467 Is Required for Smad2-Smad4 Complex Formation and Signaling* , 1997, The Journal of Biological Chemistry.

[10]  J. Kuriyan,et al.  Structures of Src-family tyrosine kinases. , 1997, Current opinion in structural biology.

[11]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[12]  J. Massagué,et al.  The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. , 1997, Genes & development.

[13]  S. Willis,et al.  Formation and activation by phosphorylation of activin receptor complexes. , 1996, Molecular endocrinology.

[14]  Kathleen R. Cho,et al.  DPC4 gene in various tumor types. , 1996, Cancer research.

[15]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[16]  M. Sporn,et al.  The Transforming Growth Factor-βs , 1991 .

[17]  J Kuriyan,et al.  The TGF beta receptor activation process: an inhibitor- to substrate-binding switch. , 2001, Molecular cell.

[18]  J. Schlessinger.  Cell Signaling by Receptor Tyrosine Kinases , 2000, Cell.

[19]  Yigong Shi,et al.  Structural insights on Smad function in TGFβ signaling , 2001 .

[20]  J. Massagué,et al.  GS domain mutations that constitutively activate T beta R‐I, the downstream signaling component in the TGF‐beta receptor complex. , 1995, The EMBO journal.

[21]  B. Qin,et al.  Crystal structure of a transcriptionally active Smad4 fragment. , 1999, Structure.

[22]  Takeshi Imamura,et al.  Smad proteins exist as monomers in vivo and undergo homo‐ and hetero‐oligomerization upon activation by serine/threonine kinase receptors , 1998, The EMBO journal.

[23]  T. Muir,et al.  Peptide ligation and its application to protein engineering. , 1999, Chemistry & biology.

[24]  Liliana Attisano,et al.  SARA, a FYVE Domain Protein that Recruits Smad2 to the TGFβ Receptor , 1998, Cell.

[25]  Irene L Andrulis,et al.  MADR2 Maps to 18q21 and Encodes a TGFβ–Regulated MAD–Related Protein That Is Functionally Mutated in Colorectal Carcinoma , 1996, Cell.

[26]  J. Wrana,et al.  The Smad pathway. , 2000, Cytokine & growth factor reviews.

[27]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[28]  R. Derynck,et al.  A kinase subdomain of transforming growth factor‐β (TGF‐β) type I receptor determines the TGF‐β intracellular signaling specificity , 1997 .

[29]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[30]  C. Wernstedt,et al.  Phosphorylation of Ser465 and Ser467 in the C Terminus of Smad2 Mediates Interaction with Smad4 and Is Required for Transforming Growth Factor-β Signaling* , 1997, The Journal of Biological Chemistry.

[31]  Takeo Iwama,et al.  Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis , 1999, Oncogene.

[32]  Kohei Miyazono,et al.  TGF-β signalling from cell membrane to nucleus through SMAD proteins , 1997, Nature.

[33]  S. Srinivasula,et al.  Structural Basis of Caspase-7 Inhibition by XIAP , 2001, Cell.

[34]  J. Schlessinger,et al.  Signaling by Receptor Tyrosine Kinases , 1993 .

[35]  Michael J. Eck,et al.  Three-dimensional structure of the tyrosine kinase c-Src , 1997, Nature.

[36]  J. Massagué.  TGF-beta signal transduction. , 1998, Annual review of biochemistry.

[37]  Yigong Shi,et al.  A structural basis for mutational inactivation of the tumour suppressor Smad4 , 1997, Nature.

[38]  S. Becker,et al.  Three-dimensional structure of the Stat3β homodimer bound to DNA , 1998, Nature.

[39]  J. Darnell.  STATs and gene regulation. , 1997, Science.

[40]  Morgan Huse,et al.  Crystal Structure of the Cytoplasmic Domain of the Type I TGF β Receptor in Complex with FKBP12 , 1999, Cell.

[41]  D Cowburn,et al.  Modular peptide recognition domains in eukaryotic signaling. , 1997, Annual review of biophysics and biomolecular structure.

[42]  Benoy M. Chacko,et al.  The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization , 2001, Nature Structural Biology.

[43]  J. Massagué,et al.  Smad4/DPC4 Silencing and Hyperactive Ras Jointly Disrupt Transforming Growth Factor-β Antiproliferative Responses in Colon Cancer Cells* , 1999, The Journal of Biological Chemistry.

[44]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[45]  Scott E. Kern,et al.  DPC4, A Candidate Tumor Suppressor Gene at Human Chromosome 18q21.1 , 1996, Science.

[46]  J. Massagué,et al.  The nuclear import function of Smad2 is masked by SARA and unmasked by TGFb-dependent phosphorylation , 2000, Nature Cell Biology.

[47]  J. Massagué,et al.  Distinct Oligomeric States of SMAD Proteins in the Transforming Growth Factor-β Pathway* , 2000, The Journal of Biological Chemistry.

[48]  Anita B. Roberts,et al.  Peptide Growth Factors and Their Receptors I , 1990, Springer Study Edition.

[49]  Yigong Shi,et al.  The L3 loop: a structural motif determining specific interactions between SMAD proteins and TGF‐β receptors , 1998, The EMBO journal.

[50]  Arthur J. Rowe,et al.  Analytical ultracentrifugation in biochemistry and polymer science , 1992 .

[51]  J. Massagué,et al.  Controlling TGF-β signaling , 2000, Genes & Development.

[52]  L. Baudhuin,et al.  Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1 , 2000, Nature Cell Biology.