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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Identification of heterogeneous ribonucleoprotein A1 as a novel substrate for protein kinase C zeta
M M Municio1, J Lozano, P Sánchez
1Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Canto Blanco, Spain.
Abstract:
The zeta isoform of protein kinase C (zeta PKC) has been shown to be an important step in mitogenic signal transduction. Using a yeast interaction screen to search for potential novel substrates of zeta PKC, we identified the heterogeneous ribonucleoprotein A1 (hnRNPA1). This protein specifically interacts with the catalytic domain of zeta PKC but not with its regulatory region or with the full-length protein, or with a kinase-defective mutant of the zeta PKC catalytic domain. In addition, no interaction was detected with other kinases such as Raf-1 or Mos, that, like zeta PKC, are critically involved in signal transduction, or with the catalytic domain of epsilon PKC, which is the PKC isotype with the highest homology to zeta PKC. hnRNPA1 is directly phosphorylated by both recombinant and native zeta PKC, and this phosphorylation is increased when zeta PKC is immunoprecipitated from mitogen-activated fibroblasts. As an additional control, hnRNPA1 is not phosphorylated appreciably by catalytic epsilon PKC or by a mixture of highly purified classical PKC isotypes maximally activated by phosphatidylserine and Ca2+. Treatment of quiescent cell cultures with a potent mitogen such as platelet-derived growth factor promotes a significant phosphorylation of hnRNPA1 in vivo that is impaired by expression of a dominant negative mutant of zeta PKC. Furthermore, expression of a catalytically active zeta PKC mutant phosphorylates hnRNPA1 in vivo. These findings suggest that zeta PKC could be critically involved in a novel pathway that connects membrane signaling to nuclear regulatory events, at the level of RNA transport and processing. Results also shown here by using different zeta PKC mutants suggesting the control of the cytoplasmic localization of hnRNPA1 by zeta PKC. Also of potential functional relevance are the results demonstrating that the phosphorylation by zeta PKC severely impairs both hnRNPA1 RNA binding and its ability to promote strand annealing in vitro.
Insights
Protein kinase C zeta (PKCζ) phosphorylates heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), impacting RNA binding and cellular localization. This suggests a novel signaling pathway connecting membrane events to nuclear RNA processing.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Protein kinase C zeta (PKCζ) is crucial in mitogenic signal transduction pathways.
- Identifying novel substrates of PKCζ is key to understanding its role in cellular signaling.
Purpose of the Study:
- To identify novel substrates of PKCζ using a yeast interaction screen.
- To investigate the interaction and functional consequences of PKCζ on heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).
Main Methods:
- Yeast interaction screen to identify PKCζ interacting proteins.
- In vitro and in vivo phosphorylation assays using recombinant and native proteins.
- Analysis of hnRNPA1 RNA binding and strand annealing capabilities.
- Mutant analysis to assess the role of PKCζ in hnRNPA1 localization.
Main Results:
- Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was identified as a specific interactor of the catalytic domain of PKCζ.
- PKCζ directly phosphorylates hnRNPA1 in vitro and in vivo, a process enhanced by mitogenic stimulation.
- Phosphorylation by PKCζ impairs hnRNPA1's RNA binding and strand annealing activities.
- PKCζ influences the cytoplasmic localization of hnRNPA1.
Conclusions:
- PKCζ is involved in a novel signaling pathway linking membrane events to nuclear RNA transport and processing.
- PKCζ-mediated phosphorylation of hnRNPA1 regulates its function in RNA metabolism.
- This interaction highlights a new mechanism for controlling gene expression at the post-transcriptional level.
