Zelluläre und biophysikalische Studien an DYRK 3 - der N-Terminus als Schlüssel zum Verständnis dieser Protein-Kinase [article]

Jonathan Wolf Müller, Martin-Luther Universität, Universitäts- Und Landesbibliothek Sachsen-Anhalt
Dual specificity tyrosine phosphorylation regulated kinase 3 (DYRK 3) is a member of the DYRK family of serine/threonine protein kinases implied in growth and developmental regulation. Within the haematopoietic system, DYRK 3 plays a unique role among the DYRK family members as line specific suppressor of red blood cell development in man and mouse. DYRK 3 consists of a central kinase domain with an associated 20 N-terminal amino acids only conserved within the DYRK family of protein kinases
more » ... protein kinases (DH box), a short C-terminal part and a large amino acids spanning N‑terminal domain with unknown function (184 or 164 amino acids in DYRK 3-Long (L) and Short (S), respectively). However, DYRK 3 action is still poorly understood. To localize DYRK 3 within the cell, protein fusions with GFP or FLAG tag were transiently transfected into different mammalian cell lines. By fluorescence microscopy and cell fractionation studies DYRK 3-S with N-terminal GFP fusion was shown to be present within the cytosol and the nucleus. As the GFP-DYRK 3-S fusion would be restricted to the cytosol simply by its size of 91.4 kDa, the protein has to be able to shuttle between cytosol and nucleus. In Contrast, a fusion of DYRK 3 with C-terminal GFP or N-terminal FLAG tag was excluded from the nucleus of HEK 293 cells. Deletion of the N-terminal domain up to the start of the DH box caused the protein DYRK 3-S164-568-GFP to shuttle again. The N-terminal domain with C‑terminal GFP tag was excluded from the nucleus in many of the cells. Exclusion of DYRK 3-S-GFP was also clearly seen in HEP-G2 cells but not in LLC-PK1 or COS-7 cells. These findings can be explained by cell type specific cytosolic anchorage of DYRK 3 via its N-terminus. Yeast two-hybrid screens were performed to identify interacting partners of DYRK 3. Among others, one DYRK 3 interacting candidate was STAT 5A. The interaction of DYRK 3 with STAT 5A was dissected using the yeast system. Not only was the kinase activity of DYRK 3 dispensable for this interaction, but the N-terminal frag [...]
doi:10.25673/3495 fatcat:6mjph5o425hnphynwpgbx7cvki