Pawel Borowicz

• Borowicz, Pawel; Gjerstad, Vibeke Sundvold; Chan, Hanna; Abrahamsen, Greger; Kjelstrup, Hanna & Nyman, Tuula Anneli [Show all 7 contributors for this article] (2021). Tyr192 Regulates Lymphocyte-Specific Tyrosine Kinase Activity in T Cells. Journal of Immunology. ISSN 0022-1767. 207(4), p. 1128–1137. doi: 10.4049/jimmunol.2001105. Show summary

  TCR signaling critically depends on the tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase). Two phosphotyrosines, the activating pTyr394 and the inhibitory pTyr505, control Lck activity. Recently, pTyr192 in the Lck SH2 domain emerged as a third regulator. How pTyr192 may affect Lck function remains unclear. In this study, we explored the role of Lck Tyr192 using CRISPR/Cas9-targeted knock-in mutations in the human Jurkat T cell line. Our data reveal that both Lck pTyr394 and pTyr505 are controlled by Lck Tyr192. Lck with a nonphosphorylated SH2 domain (Lck Phe192) displayed hyperactivity, possibly by promoting Lck Tyr394 transphosphorylation. Lck Glu192 mimicking stable Lck pTyr192 was inhibited by Tyr505 hyperphosphorylation. To overcome this effect, we further mutated Tyr505. The resulting Lck Glu192/Phe505 displayed strongly increased amounts of pTyr394 both in resting and activated T cells. Our results suggest that a fundamental role of Lck pTyr192 may be to protect Lck pTyr394 and/or pTyr505 to maintain a pool of already active Lck in resting T cells. This provides an additional mechanism for fine-tuning of Lck as well as T cell activity.

• Borowicz, Pawel; Chan, Hanna; Hauge, Anette & Spurkland, Anne (2020). Adaptor proteins: Flexible and dynamic modulators of immune cell signalling. Scandinavian Journal of Immunology. ISSN 0300-9475. 92(6). doi: 10.1111/sji.12951. Full text in Research Archive Show summary

  To maintain homeostasis, all cells respond to environmental cues via a multitude of surface receptors. In order to act appropriately in their environment, cells are dependent on the transduction of the incoming signal through tightly regulated and interconnected signalling pathways to the cell nucleus. In particular, cells implicated in the immune system greatly depend on such systems to respond in a flexible and dynamic manner to environmental challenges. One major group of intracellular proteins that are involved in these signalling pathways are adaptor proteins. Although adaptor proteins are essential for normal immune cell operation, the functional role of this group of signalling proteins remains to be fully appreciated. So far, research on adaptor proteins has revealed their unique potential in building transient complexes in a reversible, dynamic and inducible manner. In this review, we explore the roles of adaptor proteins – in space and time of intracellular signalling – and their associations with human disease. Examples of adaptor proteins expressed in hematopoietic cells highlight their crucial role in the immune system. Lastly, we present challenges faced in elucidating roles of adaptor proteins, as illustrated by the T cell–specific adaptor (TSAd) protein encoded by the SH2D2A gene.

• Borowicz, Pawel; Chan, Hanna; Talmo Medina, Daniel; Gumpelmair, Simon; Kjelstrup, Hanna & Spurkland, Anne (2019). A simple and efficient workflow for generation of knock-in mutations in Jurkat T cells using CRISPR/Cas9. Scandinavian Journal of Immunology. ISSN 0300-9475. doi: 10.1111/sji.12862. Full text in Research Archive

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• Visser, Johan Georg; Borowicz, Pawel; Chan, Hanna; Hauge, Anette; Kjelstrup, Hanna & Spurkland, Anne (2023). Lck-SH2 domain superbinder for characterization of phosphotyrosine signaling in T-cell receptor pathway.
• Borowicz, Pawel; Peters, Timo; Platzer, Rene; Seigner, Jacqueline; el Darwich, Manal & Kjelstrup, Hanna [Show all 9 contributors for this article] (2023). Chimeric antigen receptors with Lck-adaptors' sequences.
• Gilmour, Brian Christopher; Chan, Hanna; Borowicz, Pawel; Phuyal, Santosh; Lossius, Andreas & Spurkland, Anne (2023). β2, or not β2? Connecting the Lck-adaptor TSAd to integrin β2 via a combined wet lab & bioinformatic approach.
• Gilmour, Brian Christopher; Chan, Hanna; Borowicz, Pawel; Johan Georg, Visser; Lossius, Andreas & Spurkland, Anne (2022). Applying bioinformatics to pick-apart elusive protein functions: Focusing on TSAd/SH2D2A, an immune kinase adaptor.
• Chan, Hanna; Borowicz, Pawel; Kjelstrup, Hanna; Gilmour, Brian Christopher; Loza, Ivan Garcia & Stensland, Maria [Show all 8 contributors for this article] (2022). Novel interaction partners of the SH2 domain in T cell specific adaptor protein (TSAd).
• Borowicz, Pawel; Gopalakrishnan, Ramakrishna Prabhu; Chan, Hanna; Granum, Stine; Abrahamsen, Greger & Foss, Stian [Show all 9 contributors for this article] (2022). How to investigate the function of a single amino acid? Using the example of a conserved pTyr motif in the unstructured C-terminus of TSAd.
• Gilmour, Brian Christopher; Chan, Hanna; Borowicz, Pawel; Kjelstrup, Hanna & Spurkland, Anne (2021). Application of bioinformatics to outstanding questions in immunology: TSAd (T cell adapter protein).
• Borowicz, Pawel; Gopalakrishnan, Ramakrishna Prabhu; Chan, Hanna; Kjelstrup, Hanna; Foss, Stian & De Souza, Gustavo Antonio [Show all 8 contributors for this article] (2019). A conserved pTyr motif in the unstructured C-terminus regulates the function of the T cell specific adaptor TSAd.
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Chan, Hanna; Abrahamsen, Greger; Kjelstrup, Hanna & Medina, Daniel [Show all 8 contributors for this article] (2019). Tyr192 in the Lck SH2 domain affects the regulation of Lck activity.
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger; Kjelstrup, Hanna; Nyman, Tuula Anneli & Spurkland, Anne (2018). Lck’s interactome is influenced by phosphorylation of Tyr192 in the Lck SH2 domain.
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger; Kjelstrup, Hanna; Nyman, Tuula Anneli & Spurkland, Anne (2018). Lck’s interactome is influenced by phosphorylation of Tyr192 in the Lck SH2 domain.
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger; Kjelstrup, Hanna; Nyman, Tuula Anneli & Spurkland, Anne (2018). Lck’s interactome is influenced by phosphorylation of Tyr192 in the Lck SH2 domain.
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger; Kjelstrup, Hanna & Spurkland, Anne (2017). The regulatory role of Lck Tyr192 in T cell receptor signalling.
• Borowicz, Pawel; Gopalakrishnan, Ramakrishna Prabhu; De Souza, Gustavo Antonio; Abrahamsen, Greger; Sundvold-Gjerstad, Vibeke & Spurkland, Anne (2017). The interactome of TSAd phosphopeptides in T cells.
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger & Spurkland, Anne (2016). Generation of CRISPR/Cas9 mutants in order to study TCR signalling pathway.
• Borowicz, Pawel & Spurkland, Anne (2016). Intracellular signaling in CRISPR mutated Jurkat T cells.
• Borowicz, Pawel & Spurkland, Anne (2020). Regulation of T cell activation by two conserved phosphotyrosines in Lck and its adapter protein TSAd. Universitetet i Oslo. ISSN 978-82-8377-575-4. Full text in Research Archive Show summary

  T cells are major players in the immune system. These cells can recognize dangerous antigens through their specific receptors. Harnessing the power of T cells would be beneficial for not only fighting numerous infectious diseases, but also for treating cancer or preventing autoimmune diseases. However, the intracellular signalling involved in the T-cell receptor (TCR) pathway is remarkably complex. It is necessary to understand it more fully in order to control the T cells behaviour. We focused on two proteins involved in TCR signalling. First, lymphocyte-specific protein tyrosine kinase (Lck) starts a phosphorylation cascade downstream of TCR stimulation. Lck itself is tightly controlled by the phosphorylation of two tyrosines: Tyr394 and Tyr505. Recently a third tyrosine, Tyr192, emerged as an important regulatory site. Second, T cell specific adaptor protein (TSAd) is best known for its interaction with Lck. Its main function is to facilitate binding between other proteins. TSAd itself contains a number of phosphotyrosines. One of the most interesting is Tyr290, as it is highly conserved, but it is not predicted to be a prototypic binding site. The aim of this thesis was to understand the function of two phosphotyrosines, Tyr192 in Lck and Tyr290 in TSAd, in order to better comprehend the intracellular signalling of the TCR. The study was based on the knock-in cell lines generated with CRISPR/Cas9 genome editing. The mutated cell lines were studied using a wide spectrum of molecular biology techniques, including mass spectrometry, western blotting, immunoprecipitation and flow cytometry. Our findings revealed that Lck Tyr192 could control and stabilize Lck activity, both positively and negatively. TSAd Tyr290 could also control the protein’s activity. Our results suggest that this tyrosine most likely facilitates the proteins conformation. We have shown that both phosphotyrosines could influence the whole TCR signalling pathway.

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