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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/213429
Regulation of CK2 by phosphoinositides and inositol pyrophosphates: implications in endocytosis
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[eng] CK2 is a constitutively active serine/threonine kinase that consists in a tetramer composed by two catalytic and two regulatory subunits (termed Cka1, Cka2, Ckb1 and Ckb2 in yeast, respectively). Catalytic subunits drive the enzyme activity independently of regulatory subunits, which stabilize the tetramer and serve as a docking platform for the majority of targets. CK2 is vital for cell viability, involved in processes like cell growth, apoptosis or stress responses, yet its dysregulation is associated with diseases, particularly cancer. Increase activity and expression of catalytic subunits along with a downregulation in regulatory ones has been described to promote tumor progression. However, little is known about the regulation of the two CK2 isoforms (tCK2 and mCK2) and the biological consequences of its alteration. Previous research indicates that binding to the phosphoinositide PI(4,5)P2 leads to tCK2 disassembly and inactivation. During the thesis we delve into the mechanism by which PI(4,5)P2 (or other phosphoinositides) regulate the balance between tCK2 and mCK2 and, in turn, understand how they regulate different cellular processes, with particular attention to endocytosis. We were able to define the existence of a lipid binding pocket in Cka1 that resides close to the interface between catalytic and regulatory subunits, in a key region for the constitutive activity of CK2. Modelling the cavity we observed that it can accommodate phosphoinositides such as PI(4,5)P2 and PI4P and inositol polyphosphates as I(1,4,5)P3 or 5-IP7. Structural analysis revealed that Cka1 residues K75-78 bind preferentially to phosphates in position 5 from I(1,4,5)P3 or PI(4,5)P2, while R81, R194, N228 and R230 bind to phosphates located in positions 4 from I(1,4,5)P3, PI(4,5)P2, PI4P or 5 in 5-IP7. The mutation of residues R81, R194, N228, R230 (main) of Cka1 and Cka2 caused their disappearance in the cytosol after fractionations, probably by inducing their aggregation and degradation. We observed that binding to PI4P through R81, R194, N228 and R230 promoted the accumulation of mCK2, as indicated by the native PAGE results from Cka1-main mutant and after depleting the kinases and phosphatases involved in PI4P metabolism. On the contrary, results from Kcs1, the kinase responsible for the pyrophosphate 5-IP7, revealed that the binding of 5-IP7 to these same residues promoted the assembly of tCK2 probably by producing a conformational change in Cka1 β4/β5 loop. Furthermore, we found that the mCK2 isoform, elevated in kcs1∆ and ckb1∆ ckb2∆ mutants, accelerated the dynamics of Myo5, Sla1, Sla2, Ede1, reducing their time anchored to the plasma membrane during endocytosis. These mutants also exhibit higher rates of endocytic events per minute compared to WT cells. Conversely, in the Cka1-main mutant with increased tCK2, the opposite effect is observed, with prolonged lifespans of endocytic complexes and lower endocytic event ratio. Treatment with the CK2 inhibitor TBB, recovered WT dynamics in all of these mutants, indicating that both isoforms were able to phosphorylate endocytic coat components producing different outcomes. Finally, we conducted a phosphoproteome to identify other functional nodes in which mCK2 participated in addition to endocytosis. Results from mCK2 (ckb1∆ ckb2∆) revealed a pool of proteins downregulated, corresponding to exclusive targets of tCK2, but also a pool of upregulated proteins, which may correspond to targets exclusively phosphorylated by mCK2. Gene ontology analysis revealed that both downregulated and upregulated proteins were associated to same processes, being ribosome biogenesis more significant in the upregulated proteins and chromatin organization in the downregulated ones. Given that both groups shared some proteins and processes we hypothesize that, similar to endocytosis, the CK2 isoforms may be controlling the dynamics of protein complexes associated to these processes.
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PASTOR GONZÁLEZ, Laura. Regulation of CK2 by phosphoinositides and inositol pyrophosphates: implications in endocytosis. [consulta: 24 de novembre de 2025]. [Disponible a: https://hdl.handle.net/2445/213429]