Biol403 - Receptor Serine/Threonine Kinases The TGFβ (transforming growth factorβ) family of growth factors TGFβ1 was first identified as a transforming factor; however, it is a member of a family of structurally related proteins, most of which have nothing to do with transformation. They have roles in regulating cell proliferation, differentiation, migration and apoptosis. Loss of function mutations in the related genes in Drosophila and Xenopus give rise to developmental defects. It was anticipated that these receptors would be linked, either directly, or indirectly, to tyrosine phosphorylation events. However, receptors for TGFβ1 contain ser/thr-protein kinase domains. TGFα is a totally unrelated growth factor that, despite its name, acts conventionally through a tyrosine kinase pathway. Human Bone Morphogenetic Protein-2 (Bmp-2) 1
The TβR family of receptors This family of receptors are divided into two subfamilies, type I and type II. These are very similar, both receptors are glycoproteins with molecular weights of 55 kda and 70 kda respectively. Both have a single membrane-spanning segment and an intrinsic ser/thr protein kinase domain in the C-terminal segment. The type I receptor has a highly conserved region of 30 amino acids, immediately preceding the protein kinase domain, containing the sequence TTSGSGSGLP. This GS domain becomes phosphorylated after binding of TGFβ. Importantly, the protein kinase activity of TβR-II is constitutively active. Domain structure Activin recpt. (21->114); transmemb. (126->148); GS (175->205); STYKc (205- >471). >ALK-5 MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTET TDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGP VELAAVIAGPVCFVCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMT TSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFRE AEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIK LALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTI DIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQ LPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTA LRIKKTLSQLSQQEG IKM TGFβ receptor type I precursor - ALK-5 2
Domain structure Transmembrane (167->189); STYKc (244->539). >TGFBR2 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPL GVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANN INHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDI FSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLG SSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVD DLANSGQVGTARYMAPEVLESRMNLENAESFKQTDVYSMALVLWEMTSRCNAVGEV KDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDP EARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK TGFβ receptor type II precursor Receptor Activation TGFβ1 is a disulphide-linked homodimer that brings together pairs of type I and type II receptors to form heterotetrameric receptor complexes. The most likely sequence of events is that TGFβ1 first binds to TβR-II, then subsequently alters its conformation so that it can be recognized by TβR-I (i.e. sequential binding). When the ligand brings the two receptors into close proximity, the type II receptor, which is constitutively active, phosphorylates the three serine and two threonine residues in the GS domain of TβR-I. With this, the dormant serine-threonine protein kinase of TβR-I becomes activated. 3
Accessory receptors Type III receptors differ from type I and type II in that their intracellular domains lack any sequence motif that could be involved in signal transduction. These accessory receptors (e.g. betaglycan and endoglin) appear to modulate signalling activity at type II receptors. Transforming growth factor, β receptor III (betaglycan, 300kDa) - TGFBR3 Signal peptide (1->21); ZP domain (453- >73); transmembrane domain (785->807); low complexity domain (834->848). ZP domain -Zona pellucida domain. A large domain, containing ~ 260 amino acids. Seen in a variety of receptor-like eukaryotic glycoproteins. These proteins have a large extracellular region followed by either a transmembrane region and a very short cytoplasmic region or by a GPI-anchor. The ZP domain is located in the C-terminal portion of the extracellular region, and contains 8 conserved Cys residues, probably involved in disulphide bond formation. Ligand/Receptor combinations 4
Intracellular signalling form TβRs The downstream signalling pathway from the TβRs was revealed by searching for mammalian homologues of their counterparts in Drosophila and C. elegans. These pathways are similar to those involved in the activation of STATS through the tyrosine kinase-containing receptors such as those for interferon and erythropoietin (see Biol220 lecture notes). In brief, a receptor complex phosphorylates a transcription factor. This forms an oligomeric complex that translocates to the nucleus to interact with DNA-response elements in promoters of responsive genes. Drosophila and C. elegans Signalling Proteins Genetic screening of Drosophila and C. elegans has provided crucial information about the mechanism of TGFβ signalling in mammalian cells. A Drosophila gene - decapentaplegic (dpp), is involved in dorsal/ventral polarity, it defines the position of future limbs, including wings, legs and antennae. 5
Mutation causes pattern deficiencies and duplications. The Dpp protein acts through three receptors that are homologous to the family of mammalian TGFβ receptors; thick veins, saxophone (TβR-I) and punt (TβR-II). There is a further gene - mad (mothers against Dpp), mutation of which gives similar defects to those of dpp mutants. However wt dpp cannot restore the defects induced by mutations in mad - placing the Mad protein downstream of Dpp protein. Sequencing revealed that Mad (fly) and Sma (worm) proteins are homologous furthermore nine human homologues coding for Smad proteins (amalgamation of Sma and Mad) were revealed. Smads The Smad proteins have two regions of homology, MH1 and MH2 (Mad homology) at the N- and C-terminals. The 9 human Smads are divided into 3 groups: Receptor-regulated Smads (Smad1, 2, 3, 5 & 8) These are phsphorylated by the activated type-i receptors. Serine phosphoryation in the C-terminal MH2 domain allows these Smads to bind Smad4, subsequently they accumulate in the nucleus and interact with DNA binding proteins. Common mediator Smad4 This is required to form the hetero-oligomeric complexes with the receptor-regulated Smads. Since it lacks the consensus sequence (SXS) it is not phosphorylated by any of the receptors. It forms complexes with phosphorylated Smad1, 2, 5 and 8 that translocate to the nucleus where they act as transcription factors. Antagonistic Smads (Smad6 and Smad7) These also lack the SXS phosphorylation site and possess only a distantly related MH1-domain. When bound to TβR-1 they prevent phosphoryation of Smads 1, 2, 3, 5 and 8 i.e. they inhibit the responses to the TGFβ family. Since expression of Smad7 is induced by TGFβ1 it may form part of a feedback loop. 6
Smad domain structure. The recruitment of Smad2 and Smad3 to the TGFβ receptor complex is controlled by a membraneassociated protein SARA (Smad anchor for receptor activation). SARA presents Smad2 and Smad3 to the activated type 1 receptor by binding cooperatively to both nonphosphorylated Smads and the TGFβ receptor. Following phosphorylation of Smad2 and Smad3, the R-Smads and SARA dissociate from the TGFβ receptor complex. Phosphorylation of R-Smads relieves an autoinhibitory MH1-MH2 interaction and allows R-Smads to form complexes with Smad4 through their MH2 domains, an interaction that is mutually exclusive with SARA binding. 7
Transcriptional regulation by Smads Smad complexes can bind directly to DNA at the SBE sequence CAGAC, but optimal induction of transcription requires their association with other factors or transcriptional partners. Different transcriptional partners are involved when different receptors are activated. The transcription factor OAZ (Olf-1 associated zinc finger protein, a transcriptional partner initially identified in olfactory epithelium) binds selectively to the BMP-activated Smad1/Smad4 complex whereas FAST (Forkhead activating signal transducer-1) binds to complexes of Smad2 and 3 with Smad4, activated by activin. Smad2-interacting transcription factors have a common proline-rich Smad interaction motif (Sim) which interacts with the MH2 domain of Smad2 (and Smad3) and therefore recruits active Smad complexes to DNA. Negative regulation of the TGFβ/Smad signalling pathway. Activation of MAP kinases leads to the phosphoryation of the linker regions of R-Smads this leads to inhibition of the translocation of these Smads to the nucleus. Ubiquitin-mediated degradation of BMP R-Smads through specific interaction with smad ubiquitination regulatory factor 1 (Smurf1) also down-regulates Smad signalling. 8
R-Smads are continuously being dephosphorylated in the nucleus. This allows them to dissociate from Smad4 and to be exported to the cytoplasm. If receptors are still active the R-Smads can be rephosphorylated, can form complexes with Smad4 and then relocalize in the nucleus. This cycling provides a mechanism whereby the time that the Smad complexes remain in the nucleus directly reflects the time for which the receptors remain active. Role of Smads in tumour suppression Mutations in the components of this signal transduction pathway can increase susceptibility to aberrant cell proliferation. This may ultimately lead to the formation of tumours. Smad4 was initially identified as one of the mutated or deleted genes linked to pancreatic and other carcinomas. Most of these mutations are in the MH2 domain. They either prevent trimerization of Smad4, decrease the stability of the protein or prevent its interaction with non-smad transcriptional partners. A small group of mutations are in the MH1 domain and these prevent interaction with Smad2. 9
Although type I and type II TGF-β receptors are welldefined serine-threonine kinases, TβRII undergoes autophosphorylation on three tyrosine residues: Y259, Y336, and Y424, albeit at a much lower level than autophosphorylation on serine and threonine residues. The Erk non-smad pathway. TGF-β can phosphorylate tyrosine residues on both type I and type II receptors and/or on Shc. The phosphorylated tyrosines are capable of recruiting Grb2/Sos to activate Erk through Ras, Raf, and their downstream MAPK cascades. Erk then regulates target gene transcription through its downstream transcription factors in conjunction with Smads to control EMT (epithelial to mesenchymal transition). Erk can also inhibit R-Smad activities through phosphorylation of R-Smads. 10
The PI3K/Akt non-smad pathway. TGF-β can activate PI3K and Akt, by inducing an interaction between the p85 subunit of PI3K and the receptors. The activated PI3K/Akt pathway then controls translational responses through mtor/s6k, which collaborates with Smad-mediated transcriptional responses during EMT. In the growth arrest response, TGF-β may inhibit S6K by inducing an interaction between the B subunit of PP2A and the receptors. Akt is also capable of antagonizing TGF-β-induced apoptosis and growth arrest by sequestering Smad3 in the cytoplasm and by inhibiting the activity of FoxO transcription factor. Summary The TGFβ1 family of growth factors have no effect on tyrosine phosphorylation. Receptors for the TGFβ1 family have a single membrane-spanning segment and an intrinsic ser/thr protein kinase domain in the C-terminal segment. The downstream signalling pathway from the TβRs was revealed by searching for mammalian homologues of their counterparts in Drosophila and C. elegans. A receptor complex phosphorylates a transcription factor (Smad). This forms an oligomeric complex that translocates to the nucleus to interact with promoters of responsive genes. Smad complexes bind directly to DNA at the SBE sequence CAGAC, but optimal induction of transcription requires their association with other factors or transcriptional partners. Mutations in the components of this signal transduction pathway can increase susceptibility to aberrant cell proliferation. This may ultimately lead to the formation of tumours. 11
References Zhang, Y. E. (2009) Cell Res. 19, 128-139. - Non-Smad signalling pathways. Eivers, E. et al., (2009) Cyto. Grow. Fact. Rev. 20, 357-365. integration of BMP and Wnt signalling. Matsuzaki, K. (2011) Carcinogenesis 32, 1578-1588.- Smad specificity. Massague, J. (2012) Nature Rev. Mol. Cell Biol. 13, 616-630.- TGFβ signalling a review. Bruce, D. L. & Sapkota, G. P. (2012) FEBS Letts. 586, 1897-1905.- Phosphatases in SMAD regulation. 12