PMID- 10432310
OWN - NLM
STAT- MEDLINE
DCOM- 19991020
LR  - 20181113
IS  - 0264-6021 (Print)
IS  - 0264-6021 (Linking)
VI  - 342 ( Pt 1)
DP  - 1999 Aug 15
TI  - Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and
      Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are
      modified in human cancers.
PG  - 133-41
AB  - The ability to separate the isoforms of human tumour suppressor protein p53
      expressed in insect cells using heparin-Sepharose correlates with differences in 
      the isoelectric point of p53, demonstrating that p53 can be heterogeneously
      modified and providing support for the use of insect cells as a model system for 
      identifying novel signalling pathways that target p53. One p53 isoform that was
      reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its
      binding to DO-1 by prior incubation with protein phosphatases, suggesting the
      presence of a previously unidentified N-terminal phosphorylation site capable of 
      masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53
      containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the
      phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies 
      overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18)
      (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to
      determine whether direct evidence could be obtained for novel phosphorylation
      sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20)
      detected significant phosphorylation of human p53 expressed in insect cells,
      whereas the relative proportion of p53 modified at Thr(18) was substantially
      lower. The relevance of these two novel phosphorylation sites to p53 regulation
      in human cells was made evident by the extensive phosphorylation of human p53 at 
      Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53
      status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as
      effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2)
      protein binding, indicating that the functional effects of these phosphorylation 
      events might be to regulate the binding of heterologous proteins to p53. These
      results provide evidence in vivo for two novel phosphorylation sites within p53
      at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and
      indicate that some human cancers might have amplified one or more Ser(20) and
      Thr(18) kinase signalling cascades to modulate p53 activity.
FAU - Craig, A L
AU  - Craig AL
AD  - Department of Molecular and Cellular Pathology, Ninewells Medical School,
      University of Dundee, Dundee DD1 9SY, Scotland, UK.
FAU - Burch, L
AU  - Burch L
FAU - Vojtesek, B
AU  - Vojtesek B
FAU - Mikutowska, J
AU  - Mikutowska J
FAU - Thompson, A
AU  - Thompson A
FAU - Hupp, T R
AU  - Hupp TR
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Epitopes)
RN  - 0 (Nuclear Proteins)
RN  - 0 (Peptide Fragments)
RN  - 0 (Protein Isoforms)
RN  - 0 (Proto-Oncogene Proteins)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Tumor Suppressor Protein p53)
RN  - 1114-81-4 (Phosphothreonine)
RN  - 17885-08-4 (Phosphoserine)
RN  - EC 2.3.2.27 (MDM2 protein, human)
RN  - EC 2.3.2.27 (Proto-Oncogene Proteins c-mdm2)
RN  - EC 3.1.3.2 (Phosphoric Monoester Hydrolases)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Antibodies, Monoclonal/immunology
MH  - Antibody Specificity
MH  - Breast Neoplasms/immunology/*metabolism
MH  - Cell Line
MH  - Epitopes/immunology/metabolism
MH  - Humans
MH  - Isoelectric Point
MH  - *Nuclear Proteins
MH  - Peptide Fragments/chemical synthesis/immunology/metabolism
MH  - Phosphoric Monoester Hydrolases/metabolism
MH  - Phosphorylation
MH  - Phosphoserine/immunology/*metabolism
MH  - Phosphothreonine/immunology/*metabolism
MH  - Protein Isoforms/genetics/immunology/metabolism
MH  - Protein Processing, Post-Translational
MH  - Proto-Oncogene Proteins/*metabolism
MH  - Proto-Oncogene Proteins c-mdm2
MH  - Recombinant Proteins/immunology/metabolism
MH  - Signal Transduction
MH  - Spodoptera
MH  - Tumor Suppressor Protein p53/genetics/immunology/*metabolism
PMC - PMC1220446
EDAT- 1999/08/05 00:00
MHDA- 1999/08/05 00:01
CRDT- 1999/08/05 00:00
PHST- 1999/08/05 00:00 [pubmed]
PHST- 1999/08/05 00:01 [medline]
PHST- 1999/08/05 00:00 [entrez]
PST - ppublish
SO  - Biochem J. 1999 Aug 15;342 ( Pt 1):133-41.