TABLE 1.

Investigation of the interaction between XFoxH1a FM and Smad2a

ResiduesbSmad2cAbility to be phosphorylatedComplex formationFM interactionSIM interaction
Wild type++++
Interface residuesK420ANTdNTNT
D450H++
K375A+D450HNTNTNT
K420A+D450HNTNTNT
K375A+K420A+D450HNTNTNT
C463G++
Residues located in potentialF356ANT
    hydrophobic groovesV419A+NT++
H331A+NT++
E326A+NT++
M327A+NT++
R334A+NT++
D352A+NT++
W274A++++
Y340A++++
Residues within the SIM-bindingW368A++
    hydrophobic pocketN381A+NT++
Y366A+NT+±
V373A++
Residue close to Smad/Smad interfaceV431A+++
  • a All Smad2 mutants were tested for phosphorylation by Western blotting of whole-cell extracts with the anti-phosphorylated-Smad2 antibody. The ability of the mutants to form complexes was assayed by gel filtration analysis. Peptide pulldowns were used to determine whether the Smad2 mutants could interact with the Mixer SIM and the XFoxH1a FM.

  • b Interface residues were as previously identified (42). SIM-binding hydrophobic pocket residues in the Smad2 MH2 domain were previously described (32).

  • c Mutation of F356, a residue which is buried within Smad2, resulted in a protein that was not phosphorylated and could not interact with either the FM or the SIM, suggesting that this mutation caused a severe structural change in the Smad2. E326, M327 and H331 lie in a groove that forms in the vicinity of the phosphoserine-binding pocket upon complex formation. Although mutation of N381 has no effect on SIM binding under our experimental conditions, it is thought from modeling to be involved in forming a hydrogen bond with I304 of the Mixer SIM (32).

  • d NT, not tested.