ABSTRACT
The BRCA1 gene encodes a complex protein that appears to be involved in some aspects of DNA repair, transcription, or cell cycle regulation. The phosphorylation of BRCA1 is enhanced following episodes of DNA damage or during cell cycle progression, indicating that phosphorylation may be an important regulatory mechanism. Through a yeast two hybrid assay, we found that the beta-subunit of casein kinase 2 (CK2) associated with a carboxy-terminal region of BRCA1. This association was much weaker with the same fragment bearing a missense mutation (M1775R) that has been identified in breast tumors. The interaction was also evident in Sf9 cells. Subsequent studies showed that BRCA1 was phosphorylated in vitro by CK2. An analysis by site directed mutagenesis of BRCA1 showed that in vitro phosphorylation by CK2 required a serine at aa1572. These data implicate CK2 as a potential mediator of BRCA1 activity.
Subject(s)
BRCA1 Protein/metabolism , Protein Serine-Threonine Kinases/metabolism , Alkaline Phosphatase/metabolism , Amino Acid Substitution , Animals , BRCA1 Protein/chemistry , BRCA1 Protein/genetics , Breast/enzymology , Breast/metabolism , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Casein Kinase II , Cloning, Molecular , Humans , Insecta/cytology , Insecta/genetics , Insecta/metabolism , Phosphorylation , Precipitin Tests , Protein Binding , Protein Serine-Threonine Kinases/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Serine/genetics , Serine/metabolismABSTRACT
A biochemical approach was used to identify proteins which interact with human BRCA1. Through this work, a kinase activity which co-purifies with BRCA1 has been identified. This kinase activity, which phosphorylates BRCA1 in vitro, was originally identified in Sf9 insect cells but is also present in cells of human origin including breast and ovarian carcinoma cell lines. The BRCA1 kinase activity in vitro is associated with a fragment of BRCA1 encompassing amino acids 329-435. This peptide is also phosphorylated in various human cell lines. A computer-assisted sequence analysis revealed that this peptide was a potential substrate for phosphorylation by PKA, PKC, or CKII. However, phosphorylation by these kinases could not be demonstrated in vitro indicating the presence of another kinase activity. Phosphorylation in vitro requires a minimal domain of BRCA1 encompassing amino acids 379-408. Notably, deletion of this minimal domain abolishes growth suppression by BRCA1 indicating that this domain, as well as phosphorylation within this domain, may be important for BRCA1 function.
Subject(s)
BRCA1 Protein/metabolism , Breast Neoplasms/enzymology , Amino Acid Sequence , Binding Sites , Female , Humans , Molecular Sequence Data , Phosphorylation , Tumor Cells, CulturedABSTRACT
We have developed a differential screening technique, single plate one transfer (SPOT), that allows the easy detection of mRNAs induced only 2-fold to 3-fold or less above background. As a model system, we looked at the induction of mRNA by parathyroid hormone (PTH) in ROS 17/2.8 rat osteosarcoma cells. The basis for this technique is to symmetrically spot in quadruplicate, on a single plate, a large number of potentially positive plaques obtained from a primary, conventional screen. We then do only one transfer from this plate in order that there will be minimal variability in DNA transfer. This filter is cut into symmetrical strips so that all clones are multiply represented on each strip. These strips are then hybridized with different probes. Since each strip contains an approximately identical amount of DNA per plaque, it is possible to accurately detect mRNAs that are induced only slightly above background. Additionally, the large sizes of the DNA plaques, as well as spotting each clone serially, contribute to the sensitivity of the technique.
