Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation.
Smad4, the common Smad, is central for transforming growth factor (TGF)-beta superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-beta and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-beta-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.
Roelen
Bernard A J
BA
Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Charlestown, MA 02129, USA.
Cohen
Ori S
OS
Raychowdhury
Malay K
MK
Chadee
Deborah N
DN
Zhang
Ying
Y
Kyriakis
John M
JM
Alessandrini
Alessandro A
AA
Lin
Herbert Y
HY
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D008040:Genetic Linkage; D018374:LLC-PK1 Cells; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D013552:Swine; D013912:Threonine / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D013912:Threonine; D020928:Mitogen-Activated Protein Kinases