Final Activity Report Summary - SHP-1 regulation (The regulation of SHP-1 tyrosine phosphatase in B cell antigen receptor signalling)
B cells are part of the adaptive immune system and express the B cell antigen receptor (BCR) that is responsible for the specific recognition of antigens. The BCR consists of the surface immunoglobulin and the associated Iga/Igß heterodimer. In this complex the immunoglobulin binds the ligand, i.e. the antigen, and the Iga/Igß heterodimer triggers signal transduction. B cells that are stimulated through the BCR can proliferate and differentiate to antibody secreting plasma cells. Signal transduction from the B cell antigen receptor (BCR) is regulated by the tyrosine kinase Syk and the SH2 domain containing protein tyrosine phosphatase 1 (SHP-1). The activity, molecular interactions and sub-cellular localisation of these enzymes are controlled by posttranslational modifications, such as phosphorylation, ubiquitination or oxidation. SHP-1 has two tandem SH2 domains, a phosphatase domain and a C-terminal tail. The latter part of the molecule has regulatory role and contains tyrosine and serine residues that can be phosphorylated. However, the molecular modifications and the mechanisms by which this C-terminal tail regulates the activity and sub-cellular localisation of the phosphatase are not yet clarified.
We observed in previous studies that SHP-1 was expressed not only in the cytoplasm but also in the nucleus. The C-terminal tail played an important role in this nuclear translocation since it had a functional nuclear localisation signal (NLS). The goal of this project was to get more knowledge of these modifications of the C-terminal tail of SHP-1 to gain a better understanding of its regulation in B cells and pre-B cells. This part of the molecule contained tyrosine and serine residues that could be phosphorylated, as well as a functional nuclear localisation signal (NLS). We applied the S2 Schneider cell system that allowed for a reverse genetic approach. It was therefore possible to rebuild mammalian signalling machineries in these cells and study them in an evolutionary distant cellular environment. By this method we observed that Syk could phosphorylate the tyrosines on the C-terminal tail of SHP-1. These phosphorylated tyrosines could then serve as interaction sites for other proteins. We also studied serine phosphorylation of the C-terminal tail. Several serine or threonine kinases were tested and PKCa was able to phosphorylate S591 that was very close to the NLS.
The next question was whether this serine phosphorylation had a regulatory role in the sub-cellular localisation of the molecule. Using green fluorescence fusion proteins (GFP fusion proteins) and different mutant versions we observed that this phosphorylation indeed inhibited nuclear translocation. In B cells SHP-1 was in most cases localised in the cytosol. However, it was shown in a B cell line that certain cytokine stimuli, such as IL-7 and IL-4, could induce its nuclear localisation. IL-7 has great importance as a growth factor during B cell differentiation. Therefore, the nuclear translocation was examined in bone marrow derived pre-B cell lines that grew only in the presence of IL-7. We confirmed that in pre-B cells IL-7 signalling drove SHP-1 nuclear translocation and that pre-BCR mediated activation of PKCs led to S591 phosphorylation and, subsequently, to the inhibition of nuclear translocation. The exact role of SHP-1 in the nucleus was not known. We therefore analysed nuclear interaction partners by mass spectroscopy. The data gained by this approach showed that SHP-1 interacted with many proteins that regulated cell cycle and tumorigenesis suggesting a role in regulating the IL-7 induced proliferation in the nucleus. In order to investigate the role of SHP-1 in early B cell differentiation we generated conditional knock-out mice that had the SHP-1 gene deleted only in B cells. Pre-B cell lines were made from these mice to study the role of exclusively cytoplasmic or nuclear phosphatase molecules in pre-B cell differentiation.
It could be overall stated that SHP-1 is considered to be a tumour suppressor; however the molecular details of this effect are still unknown. Further studies on the results we obtained via the proteomic analysis of SHP-1 nuclear interactions could reveal the mechanisms of its tumour suppressor ability.
We observed in previous studies that SHP-1 was expressed not only in the cytoplasm but also in the nucleus. The C-terminal tail played an important role in this nuclear translocation since it had a functional nuclear localisation signal (NLS). The goal of this project was to get more knowledge of these modifications of the C-terminal tail of SHP-1 to gain a better understanding of its regulation in B cells and pre-B cells. This part of the molecule contained tyrosine and serine residues that could be phosphorylated, as well as a functional nuclear localisation signal (NLS). We applied the S2 Schneider cell system that allowed for a reverse genetic approach. It was therefore possible to rebuild mammalian signalling machineries in these cells and study them in an evolutionary distant cellular environment. By this method we observed that Syk could phosphorylate the tyrosines on the C-terminal tail of SHP-1. These phosphorylated tyrosines could then serve as interaction sites for other proteins. We also studied serine phosphorylation of the C-terminal tail. Several serine or threonine kinases were tested and PKCa was able to phosphorylate S591 that was very close to the NLS.
The next question was whether this serine phosphorylation had a regulatory role in the sub-cellular localisation of the molecule. Using green fluorescence fusion proteins (GFP fusion proteins) and different mutant versions we observed that this phosphorylation indeed inhibited nuclear translocation. In B cells SHP-1 was in most cases localised in the cytosol. However, it was shown in a B cell line that certain cytokine stimuli, such as IL-7 and IL-4, could induce its nuclear localisation. IL-7 has great importance as a growth factor during B cell differentiation. Therefore, the nuclear translocation was examined in bone marrow derived pre-B cell lines that grew only in the presence of IL-7. We confirmed that in pre-B cells IL-7 signalling drove SHP-1 nuclear translocation and that pre-BCR mediated activation of PKCs led to S591 phosphorylation and, subsequently, to the inhibition of nuclear translocation. The exact role of SHP-1 in the nucleus was not known. We therefore analysed nuclear interaction partners by mass spectroscopy. The data gained by this approach showed that SHP-1 interacted with many proteins that regulated cell cycle and tumorigenesis suggesting a role in regulating the IL-7 induced proliferation in the nucleus. In order to investigate the role of SHP-1 in early B cell differentiation we generated conditional knock-out mice that had the SHP-1 gene deleted only in B cells. Pre-B cell lines were made from these mice to study the role of exclusively cytoplasmic or nuclear phosphatase molecules in pre-B cell differentiation.
It could be overall stated that SHP-1 is considered to be a tumour suppressor; however the molecular details of this effect are still unknown. Further studies on the results we obtained via the proteomic analysis of SHP-1 nuclear interactions could reveal the mechanisms of its tumour suppressor ability.