Showing papers by "Kavita Shah published in 2001"

ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation

Abstract: Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is one of a family of 20 proteins that are involved in transcription and post-transcriptional messenger RNA metabolism. The mechanisms that underlie regulation of hnRNP-K activities remain largely unknown. Here we show that cytoplasmic accumulation of hnRNP-K is phosphorylation-dependent. Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) efficiently phosphorylates hnRNP-K both in vitro and in vivo at serines 284 and 353. Serum stimulation or constitutive activation of ERK kinase (MEK1) results in phosphorylation and cytoplasmic accumulation of hnRNP-K. Mutation at ERK phosphoacceptor sites in hnRNP-K abolishes the ability to accumulate in the cytoplasm and renders the protein incapable of regulating translation of mRNAs that have a differentiation-control element (DICE) in the 3' untranslated region (UTR). Similarly, treatment with a pharmacological inhibitor of the ERK pathway abolishes cytoplasmic accumulation of hnRNP-K and attenuates inhibition of mRNA translation. Our results establish the role of MAPK/ERK in phosphorylation-dependent cellular localization of hnRNP-K, which is required for its ability to silence mRNA translation.

Phage-display evolution of tyrosine kinases with altered nucleotide specificity †

Abstract: The problem of identifying downstream targets of kinase phosphorylation remains a challenge despite technological advances in genomics and proteomics. A recent approach involves the generation of kinase mutants that can uniquely use "orthogonal" ATP analogs to phosphorylate substrates in vivo. Using structure-based design, mutants of several protein kinase superfamily members have been found; robust and general methods are needed, however, for altering the nucleotide specificity of the remaining kinases in the genome. Here we demonstrate the application of a new phage display technique for direct functional selection to the identification of a tyrosine kinase mutant with the ability to use N6-benzyl-ATP. Our method produces, in five rounds of selection, a mutant identical to the best orthogonal Src kinase found to date. In addition, we isolate from a larger library of kinase mutants a promiscuous clone capable of using many different ATP analogs. This approach to engineering orthogonal kinases, combined with others, will facilitate the mapping of phosphorylation targets of any kinase in the genome.

Probing Tat peptide-TAR RNA interactions by psoralen photo-cross-linking.

Abstract: Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem−loop structure located at the 5‘-end of all HIV mRNAs. We have used a site-specific cross-linking method based on psoralen photochemistry to determine the effect of core residues from the Tat sequence on the protein orientation in the Tat−TAR complex and on the specificity of Tat−TAR binding. We synthesized two Tat fragments, Tat(42−72) and Tat(37−72), and incorporated a psoralen-modified amino acid at position 41 during solid-phase assembly of the peptides. We used these psoralen−Tat conjugates to form specific complexes with TAR RNA. Upon near-ultraviolet irradiation (360 nm), psoralen−Asp41−Tat(37−72) cross-linked to a single site in the TAR RNA sequence. The RNA−protein complex was purified and the cross-link site on TAR RNA was determined by primer extension analysis, which revealed that Asp41 of Tat is close to U42 of the lower...