• Register
X
Forgot Password

If you have forgotten your password you can enter your email here and get a temporary password sent to your email.

X

Leaving Community

Are you sure you want to leave this community? Leaving the community will revoke any permissions you have been granted in this community.

No
Yes

Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 binding, transactivation and nuclear targetting.

The transcription factor, forkhead in rhabdomyosarcoma (FKHR), is phosphorylated at three amino acid residues (Thr-24, Ser-256 and Ser-319) by protein kinase B (PKB)alpha. In the present study, mutagenesis has been used to study the roles of these phosphorylation events in regulating FKHR function in transfected HEK-293 cells. We find that the overexpression of FKHR[S256A] (where Ser-256-->Ala) blocks PKB activity in cells, preventing phosphorylation of the endogenous substrates FKHRL1 and glycogen synthase kinase-3. Thus some reported effects of overexpression of this and other mutants may be indirect, and result from suppression of the phosphorylation of other sites on FKHR and/or other PKB substrates. For example, we have shown that Thr-24 phosphorylation alone is critical for interaction with 14-3-3 proteins, and that the substitution of Ser-256 with an alanine residue indirectly blocks 14-3-3 protein binding by preventing the phosphorylation of Thr-24. We also found that insulin-like growth factor (IGF)-1 and serum-induced nuclear exclusion of FKHR[S256A] depends on the degree of overexpression of this mutant. Our results indicated that the interaction of FKHR with 14-3-3 proteins was not required for IGF-1-stimulated exclusion of FKHR from the nucleus. We present evidence in support of another mechanism, which depends on the phosphorylation of Ser-256 and may involve the masking of a nuclear localization signal. Finally, we have demonstrated that the failure of IGF-1 to suppress transactivation by FKHR[S256A] is not explained entirely by its failure to bind 14-3-3 proteins or to undergo nuclear exclusion. This result suggests that Ser-256 phosphorylation may also suppress transactivation by FKHR by yet another mechanism, perhaps by disrupting the interaction of FKHR with target DNA binding sites and/or the function of the transactivation domain.

Pubmed ID: 11237865

Authors

  • Rena G
  • Prescott AR
  • Guo S
  • Cohen P
  • Unterman TG

Journal

The Biochemical journal

Publication Data

March 15, 2001

Associated Grants

  • Agency: NIDDK NIH HHS, Id: DK41430

Mesh Terms

  • 14-3-3 Proteins
  • Active Transport, Cell Nucleus
  • Cell Line
  • Cell Nucleus
  • DNA-Binding Proteins
  • Forkhead Transcription Factors
  • Glutathione Transferase
  • Green Fluorescent Proteins
  • Humans
  • Insulin-Like Growth Factor I
  • Luminescent Proteins
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Phosphoserine
  • Phosphothreonine
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-akt
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Transcriptional Activation
  • Tyrosine 3-Monooxygenase