The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein recruits a diversity of molecular partners that are involved in signal transduction, transcription, RNA processing, and translation. K protein is phosphorylated in vivo and in vitro by inducible kinase(s) and contains several potential sites for protein kinase C (PKC) phosphorylation. In this study we show that K protein is phosphorylated in vitro by PKC␦ and by other PKCs. Deletion analysis and site-directed mutagenesis revealed that Ser 302 is a major K protein site phosphorylated by PKC␦ in vitro. This residue is located in the middle of a short amino acid fragment that divides the two clusters of SH3-binding domains. Mutation of Ser 302 decreased the level of phosphorylation of exogenously expressed K protein in phorbol 12-myristate 13-acetate-treated COS cells, suggesting that Ser 302 is also a site for PKC-mediated phosphorylation in vivo. In vitro, PKC␦ binds K protein via the highly interactive KI domain, an interaction that is blocked by poly(C) RNA. Mutation of Ser 302 did not alter the K protein-PKC␦ interaction in vitro, suggesting that phosphorylation of this residue alone is not sufficient to alter this interaction. Instead, binding of PKC␦ to K protein in vitro and in vivo was greatly increased by K protein phosphorylation on tyrosine residues. The ability of PKC␦ to bind and phosphorylate K protein may serve not only to alter the activity of K protein itself, but K protein may also bridge PKC␦ to other K protein molecular partners and thus facilitate molecular cross-talk. The regulated nature of the PKC␦-K protein interaction may serve to meet cellular needs at sites of active transcription, RNA processing and translation in response to changing extracellular environment.

The heterogeneous nuclear ribonucleoprotein K protein recruits a diversity of molecular partners and may act as a docking platform involved in such processes as transcription, RNA processing, and translation. We show that K protein is tyrosine-phosphorylated in vitro by Src and Lck. Treatment with H 2 O 2 /Na 3 VO 4 , which induces oxidative stress, stimulated tyrosine phosphorylation of K protein in cultured cells and in intact livers. Tyrosine phosphorylation increased binding of Lck and the proto-oncoprotein Vav to K protein in vitro. Oxidative stress increased the association of K protein with Lck and Vav, suggesting that tyrosine phosphorylation regulates the ability of K protein to recruit these effectors in vivo. Translation-based assay showed that K protein is constitutively bound to many mRNAs in vivo. Native immunoprecipitated K protein-mRNA complexes were disrupted by tyrosine phosphorylation, suggesting that the in vivo binding of K protein to mRNA may be responsive to the extracellular signals that activate tyrosine kinases. This study shows that tyrosine phosphorylation of K protein regulates K protein-protein and K protein-RNA interactions. These data are consistent with a model in which functional interaction of K protein is responsive to changes in the extracellular environment. Acting as a docking platform, K protein may bridge signal transduction pathways to sites of nucleic acid-dependent process such as transcription, RNA processing, and translation.The heterogeneous nuclear ribonucleoprotein (hnRNP) 1 K protein is a highly interactive factor (1, 2) that appears to act at multiple tiers of gene expression including transcription (3-5), translation (6, 7), and probably RNA processing (2, 8).K protein is composed of multiple domains that serve to engage a diverse group of molecular partners (1) including DNA (5, 9), RNA (10 -13), Vav (14, 15), transcriptional repressors (16, 17), and inducible kinases (14, 18 -22). While the interaction of K protein with the transcription factor CCAAT/ enhancer-binding protein ␤ is mediated by the N-terminal third of K protein (4), K protein interaction with an interleukin-1-responsive kinase is mediated by the C-terminal domain (14). The interaction of K protein with the Src family of tyrosine kinases (14), PKC␦ (21), Vav (14), and several transcriptional repressors (16, 17) is mediated by the centrally located prolinerich KI domain. It is interesting to note that TATA box-binding protein (5) binds adjacent to the KI domain. 2 K protein's modular structure and its highly interactive nature suggest that K protein acts as a docking platform or scaffold (1). The observations that K protein also interacts with RNA and DNA suggest that K protein may do so at sites of nucleic acid-dependent processes.K protein is phosphorylated in vivo in response to treatment of cells with cytokines, acute phase reactants, and other changes in the extracellular environment (21-23) indicating that K protein is coupled to signal transduction. Taken together, the biochemical and func...

A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes alpha, betaI, and betaII of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative "PKC kinase" for permissive activation. Expression of PKCalpha in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCalpha and PKCbetaII could be identified as Thr497 and Thr500, respectively. We report here that PKCdelta, contrary to PKCalpha, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Km values for two substrates are comparable to those of recombinant PKCdelta expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCalpha and PKCbetaII, respectively, is not essential for obtaining a catalytically competent conformation of PKCdelta. The mutant Ala505 can be activated and does not differ from the wild type regarding activity and several other features. Ser504 can not take over the role of Thr505 and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504 and Ala504/Ala505). These results indicate that phosphorylation of Thr505 is not required for the formation of functional PKCdelta and that at least this PKC isoenzyme differs from the isotypes alpha, betaI, and betaII regarding the permissive activation by a PKC kinase.