Molecular basis for phosphorylation-dependent, PEST-mediated protein turnover.

Proteasomal-mediated rapid turnover of proteins is often modulated by phosphorylation of PEST sequences. The E2 protein from papillomavirus participates in gene transcription, DNA replication, and episomal genome maintenance. Phosphorylation of a PEST sequence located in a flexible region accelerates its degradation. NMR analysis of a 29 amino acid peptide fragment derived from this region shows pH-dependent polyproline II and alpha helix structures, connected by a turn. Phosphorylation, in particular that at serine 301, disrupts the overall structure, and point mutations have either stabilizing or destabilizing effects. There is an excellent correlation between the thermodynamic stability of different peptides and the half-life of E2 proteins containing the same mutations in vivo. The structure around the PEST region appears to have evolved a marginal stability that is finely tunable by phosphorylation. Thus, conformational stability, rather than recognition of a phosphate modification, modulates the degradation of this PEST sequence by the proteasome machinery.

[1]  Erica S. Johnson,et al.  Methotrexate Inhibits Proteolysis of Dihydrofolate Reductase by the N-end Rule Pathway (*) , 1995, The Journal of Biological Chemistry.

[2]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[3]  A. Bax,et al.  An empirical correlation between 1JC.alpha.H.alpha. and protein backbone conformation , 1992 .

[4]  V. Hsu,et al.  NMR identification of left‐handed polyproline type II helices , 2003, Biopolymers.

[5]  Robert W Woody,et al.  Solvent dependence of PII conformation in model alanine peptides. , 2004, Journal of the American Chemical Society.

[6]  S. Rogers,et al.  PEST sequences and regulation by proteolysis. , 1996, Trends in biochemical sciences.

[7]  A. Ciechanover,et al.  Modes of regulation of ubiquitin‐mediated protein degradation , 2000, Journal of cellular physiology.

[8]  S. Grzesiek,et al.  The Importance of Not Saturating H2o in Protein NMR : application to Sensitivity Enhancement and Noe Measurements , 1993 .

[9]  M. Buck,et al.  Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins , 1998, Quarterly Reviews of Biophysics.

[10]  K. Wüthrich,et al.  Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. , 1983, Biochemical and biophysical research communications.

[11]  Luis Serrano,et al.  Elucidating the folding problem of helical peptides using empirical parameters , 1994, Nature Structural Biology.

[12]  Ad Bax,et al.  MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy , 1985 .

[13]  S. Krimm,et al.  New chain conformations of poly(glutamic acid) and polylysine. , 1968, Biopolymers.

[14]  J. Dillner,et al.  Structural analysis of the human papillomavirus type 16-E2 transactivator with antipeptide antibodies reveals a high mobility region linking the transactivation and the DNA-binding domains. , 1991, Nucleic acids research.

[15]  S. Krimm,et al.  Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride , 1973 .

[16]  R. Griffey,et al.  Correlation of proton and nitrogen-15 chemical shifts by multiple quantum NMR☆ , 1983 .

[17]  L Serrano,et al.  Elucidating the folding problem of helical peptides using empirical parameters. III. Temperature and pH dependence. , 1995, Journal of molecular biology.

[18]  A. Matouschek,et al.  ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. , 2001, Molecular cell.

[19]  A. Fersht,et al.  An N-terminal fragment of barnase has residual helical structure similar to that in a refolding intermediate. , 1992, Journal of molecular biology.

[20]  Alison A. McBride,et al.  Casein Kinase II Phosphorylation-induced Conformational Switch Triggers Degradation of the Papillomavirus E2 Protein* , 2004, Journal of Biological Chemistry.

[21]  G. Prat-Gay.  Conformational Preferences of a Peptide Corresponding to the Major Antigenic Determinant of Foot-and- Mouth Disease Virus: Implications for Peptide-Vaccine Approaches , 1997 .

[22]  Robert W Woody,et al.  Is polyproline II a major backbone conformation in unfolded proteins? , 2002, Advances in protein chemistry.

[23]  George D Rose,et al.  Polyproline II structure in a sequence of seven alanine residues , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Rechsteiner.  Natural substrates of the Ubiquitin proteolytic pathway , 1991, Cell.

[25]  M. Rechsteiner,et al.  PEST sequences are signals for rapid intracellular proteolysis. , 1990, Seminars in cell biology.

[26]  J. Warwicker,et al.  Effect of phosphorylation on α-helix stability as a function of position , 2002 .

[27]  S. Rogers,et al.  Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. , 1986, Science.

[28]  J M Thornton,et al.  Analysis of main chain torsion angles in proteins: prediction of NMR coupling constants for native and random coil conformations. , 1996, Journal of molecular biology.

[29]  N. Kallenbach,et al.  Stabilization of the ribonuclease S‐peptide α‐helix by trifluoroethanol , 1986 .

[30]  P. Howley,et al.  Bovine papillomavirus with a mutation in the E2 serine 301 phosphorylation site replicates at a high copy number , 1991, Journal of virology.

[31]  E. Stellwagen,et al.  The role of PII conformations in the calculation of peptide fractional helix content , 1997, Protein science : a publication of the Protein Society.

[32]  Y H Chen,et al.  Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. , 1974, Biochemistry.

[33]  C. Pace,et al.  Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding , 1995, Protein science : a publication of the Protein Society.

[34]  S. Krimm,et al.  Effwct of temperature on the circular dichroism spectra of polypeptides in the extended state , 1972, Biopolymers.

[35]  Richard R. Ernst,et al.  Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy , 1983 .

[36]  B D Sykes,et al.  Chemical shifts as a tool for structure determination. , 1994, Methods in enzymology.

[37]  A. McBride,et al.  Bovine Papillomavirus Type 1 Genomes and the E2 Transactivator Protein Are Closely Associated with Mitotic Chromatin , 1998, Journal of Virology.

[38]  L. Iakoucheva,et al.  The importance of intrinsic disorder for protein phosphorylation. , 2004, Nucleic acids research.

[39]  S. Shumway,et al.  The PEST Domain of IκBα Is Necessary and Sufficient forin Vitro Degradation by μ-Calpain* , 1999, The Journal of Biological Chemistry.

[40]  A. McBride,et al.  Proteasome-Mediated Degradation of the Papillomavirus E2-TA Protein Is Regulated by Phosphorylation and Can Modulate Viral Genome Copy Number , 2000, Journal of Virology.

[41]  R. Hampton.  Proteolysis and sterol regulation. , 2002, Annual review of cell and developmental biology.

[42]  A. Matouschek,et al.  An unstructured initiation site is required for efficient proteasome-mediated degradation , 2004, Nature Structural &Molecular Biology.

[43]  A. Tall,et al.  Phosphorylation of a Pest Sequence in ABCA1 Promotes Calpain Degradation and Is Reversed by ApoA-I* , 2003, Journal of Biological Chemistry.

[44]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[45]  A. Fersht,et al.  Quantitative determination of helical propensities from trifluoroethanol titration curves. , 1994, Biochemistry.

[46]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[47]  K. Anderson,et al.  Cactus protein degradation mediates Drosophila dorsal-ventral signaling. , 1995, Genes & development.

[48]  Bruce A. Johnson,et al.  NMR View: A computer program for the visualization and analysis of NMR data , 1994, Journal of biomolecular NMR.

[49]  J. Yewdell,et al.  Proteasomes get by with lots of help from their friends. , 2004, Immunity.