Abstract
The heterogeneous nuclear ribonucleoprotein particle (hnRNP) K protein is comprised of multiple modular domains that serve to engage a diverse group of molecular partners including DNA, RNA, the product of the proto-oncogene vav, and tyrosine and serine/threonine kinases. To identify additional K protein molecular partners and to further understand its function, we used a fragment of K protein as a bait in the yeast two-hybrid screen. The deduced primary structure of one of the positive clones revealed a novel zinc finger protein, hereby denoted as Zik1. In addition to the nine contiguous zinc fingers in the C terminus, Zik1 contains a KRAB-A domain thought to be involved in transcriptional repression. Zik1 and K protein bound in vitro and co-immunoprecipitated from cell extracts indicating that in vivo their interaction is direct. Expression of Gal4 DNA-binding domain-Zik1 fusion protein repressed a gene promoter bearing Gal4-binding elements, indicating that from cognate DNA elements Zik1 is a transcriptional repressor. The known diverse nature of K protein molecular interactions and now the identification of a K protein partner that is a transcriptional repressor lends support to the notion that K protein is a remarkably versatile molecule that may be acting as a docking platform to facilitate communication among molecules involved in signal transduction and gene expression.
Highlights
K protein was first discovered as a component of the heterogeneous nuclear ribonucleoprotein particle1 from which it derives its name [1, 2]
Since its original discovery in heterogeneous nuclear ribonucleoprotein particle (hnRNP), K protein has been shown to be a highly versatile molecule that engages nucleic acids and an array of proteins, it interacts in vivo and in vitro with the product of the proto-oncogene vav, the protein-tyrosine kinase, Src [14, 16, 17], and the TFIID TATAbinding protein [8]
Cloning of a Novel Protein That Interacts with hnRNP K—The N-terminal two-thirds of K protein [14] was used as a bait in two-hybrid screen to isolate K protein molecular partners
Summary
K protein was first discovered as a component of the heterogeneous nuclear ribonucleoprotein particle (hnRNP)1 from which it derives its name [1, 2]. This library was inserted into the transcription activation domain vector, pVP16, to generate VP16-cDNA hybrid proteins. The final construct contained an insert encoding the Gal4 DNA-binding domain-Zik1 fusion protein under the control of SV40 promoter.
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