Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K: implications in the regulation of SRC1A transcription.
The human SRC gene encodes pp60(c-src), a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes.
Ritchie
Shawn A
SA
Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
Pasha
Mohammed K
MK
Batten
Danielle J P
DJ
Sharma
Rajendra K
RK
Olson
Douglas J H
DJ
Ross
Andrew R S
AR
Bonham
Keith
K
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000528:radiation effects; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D009841:Oligonucleotides; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D016392:Proto-Oncogene Proteins pp60(c-src); C030986:pyrimidine