ABSTRACT
Many regulatory proteins, including the transcription factor c-Jun, are highly enriched in disordered protein regions that govern growth, division, survival, differentiation, and response to signals. The stability of c-Jun is controlled by poorly understood regulatory interactions of its disordered region with both the E3 ubiquitin ligase SCFFbw7 and prolyl cis-trans isomerase Pin1. We use nuclear magnetic resonance and fluorescence studies of c-Jun to demonstrate that multisite c-Jun phosphorylation is required for high-affinity interaction with Fbw7. We show that the Pin1 WW and PPIase domains interact in a dynamic complex with multiply phosphorylated c-Jun. Importantly, Pin1 isomerizes a pSer-Pro peptide bond at the c-Jun N terminus that affects binding to Fbw7 and thus modulates the ubiquitin-mediated degradation of c-Jun. Our findings support the general principle that multiple weak binding motifs within disordered regions can synergize to yield high-affinity interactions and provide rapidly evolvable means to build and fine-tune regulatory events.
Subject(s)
F-Box-WD Repeat-Containing Protein 7/chemistry , Intrinsically Disordered Proteins/chemistry , JNK Mitogen-Activated Protein Kinases/chemistry , NIMA-Interacting Peptidylprolyl Isomerase/chemistry , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , F-Box-WD Repeat-Containing Protein 7/genetics , F-Box-WD Repeat-Containing Protein 7/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/metabolism , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Kinetics , Models, Molecular , NIMA-Interacting Peptidylprolyl Isomerase/genetics , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Stability , Protein Structure, Secondary , Proteolysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Substrate Specificity , ThermodynamicsABSTRACT
Hierarchic phosphorylation and concomitant Pin1-mediated proline isomerization of the oncoprotein c-Myc controls its cellular stability and activity. However, the molecular basis for Pin1 recognition and catalysis of c-Myc and other multisite, disordered substrates in cell regulation and disease is unclear. By nuclear magnetic resonance, surface plasmon resonance, and molecular modeling, we show that Pin1 subdomains jointly pre-anchor unphosphorylated c-Myc1-88 in the Pin1 interdomain cleft in a disordered, or "fuzzy", complex at the herein named Myc Box 0 (MB0) conserved region N-terminal to the highly conserved Myc Box I (MBI). Ser62 phosphorylation in MBI intensifies previously transient MBI-Pin1 interactions in c-Myc1-88 binding, and increasingly engages Pin1PPIase and its catalytic region with maintained MB0 interactions. In cellular assays, MB0 mutated c-Myc shows decreased Pin1 interaction, increased protein half-life, but lowered rates of Myc-driven transcription and cell proliferation. We propose that dynamic Pin1 recognition of MB0 contributes to the regulation of c-Myc activity in cells.
Subject(s)
Peptidylprolyl Isomerase/chemistry , Proto-Oncogene Proteins c-myc/metabolism , Amino Acid Sequence , Binding Sites , Humans , Molecular Sequence Data , Mutation , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/genetics , Peptidylprolyl Isomerase/metabolism , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-myc/chemistryABSTRACT
In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251-73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.
