Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses.
Transforming growth factor-beta (TGF-beta) elicits cellular effects by activating specific Smad proteins that control the transcription of target genes. Whereas there is growing evidence that there are TGF-beta type I receptor-initiated intracellular pathways that are distinct from the pivotal Smad pathway, their physiological importance in TGF-beta signaling is not well understood. Therefore, we generated TGF-beta type I receptors (also termed ALK5s) with mutations in the L45 loop of the kinase domain, termed ALK5(D266A) and ALK5(3A). These mutants showed retained kinase activity but were unable to activate Smads. Characterization of their signaling properties revealed that the two L45 loop mutants did not mediate Smad-dependent transcriptional responses, TGF-beta-induced growth inhibition, and fibronectin and plasminogen activator-1 production in R4-2 mink lung epithelial cells lacking functional ALK5 protein. Mutation in the L45 loop region did not affect the binding of inhibitory Smads but did abrogate the weak binding of X-linked inhibitor of apoptosis protein and Disabled-2 to ALK5. This suggests that the L45 loop in the kinase domain is important for docking of other binding proteins. Interestingly, JNK MAP kinase activity was found to be activated by the ALK5(3A) mutant in various cell types. In addition, TGF-beta-induced inhibition of cyclin D1 expression and stimulation of PMEPA1 (androgen-regulated prostatic mRNA) expression were found to occur, albeit weakly, in an Smad-independent manner in normal murine mammary gland cells. However, the TGF-beta-induced epithelial to mesenchymal transdifferentiation was found to require an intact L45 loop and is likely to be dependent on the Smad pathways.
Itoh
Susumu
S
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Thorikay
Midory
M
Kowanetz
Marcin
M
Moustakas
Aristidis
A
Itoh
Fumiko
F
Heldin
Carl-Henrik
CH
ten Dijke
Peter
P
D030201:Activin Receptors, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D002455:Cell Division / Q000502:physiology; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005353:Fibronectins / Q000096:biosynthesis; D016147:Genes, Tumor Suppressor; D006801:Humans; D008907:Mink; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051785:Smad Proteins; D015534:Trans-Activators / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051017:Apoptosis Regulatory Proteins; C102885:DAB2 protein, human; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005353:Fibronectins; D017395:Plasminogen Activator Inhibitor 1; D011506:Proteins; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human