In this work I focused on functional correlations between some important protein kinases, tightly involved in cell viability. The project was divided in the following major parts: a) CK2 indirectly modulates phosphorylation state of Akt pThr308. Protein kinase CK2 is a Ser/Thr protein kinase composed of two catalytic (? and/or ?') and two regulatory (?) subunits. It is ubiquitous, constitutively active, and pleiotropic, with more than 300 protein substrates known so far. CK2 plays an antiapoptotic role, coordinating a network of signaling pathways essential for cell survival. One of these is rapresented by Akt, a kinase whose mechanism of activation is based on the phosphorylation of two key residues: Thr308 (by the PDK1 kinase) and Ser473 (by the mTOR/Rictor complex). On the other side, the dephosphorylation of Thr308 and Ser473 is accounted for by PP2A phosphatase and PHLPP, respectively. CK2 acts directly on Akt, phosphorylating its Ser129 residue, both in vitro and in vivo, thus enhancing its catalytic activity. Here we also show that downregulation of CK2 activity by inhibitors, or mutation of the CK2-dependent site in Akt (Ser129Ala), induce a lower phosphorylation of Thr308, which is not a direct target for CK2. This finding can be explained assuming that phosphorylation of Akt at Ser129 by CK2 facilitates the recognition of Thr308 by the kinase PDK1 or, alternatively, makes Akt less accessible to the phosphatase PP2A. We found that PDK1 does not discriminate between the two different forms of Akt, phosphorylated or not at the CK2 site; on the contrary, our data show that, in case of Ser129 phosphorylation, a lower degree of T308 dephosphorylation occurs. In fact, when we treat cells with the PP2A inhibitor okadaic acid, any difference at Thr308 phosphorylation is abrogated between wt Akt (phosphorylated at Ser129 by CK2) and Ser129Ala Akt mutant. We also found that Hsp90, involved in preserving the Thr308 phosphorylation state, is less tightly associated to Akt in case of the Ser129Ala mutation. This indicates that the regulation operated by CK2 on Akt can be ascribed, at least in part, to a protection from the pThr308-dephosphorylation process, possibly involving other protein partners, such as the Cdc37/Hsp90 complex. b) Drug resistance and pro-survival protein kinases. Given the pro-survival and anti-apoptotic function of CK2 and Akt, we also investigated their possible involvement in the multidrug resistance phenotype, a condition where a high proliferation rate and a reduced cell death degree result in the failure of cancer therapy. We analysed the expression level of these two kinases and their role in some cell lines, available in the two variants normally sensitive (S) or resistant (R) to chemical apoptosis. Most of the work was focused on T-lymphoblastoid cells (CEM-S and CEM-R), but we also extended our analyses to other cell models, particullary to an osteosarcoma cell line (U2OS-S and U2OS-R) and to ovarian carcinoma cells (2008-S and 2008-R). We found that CK2 and Akt are differently expressed throughout our cellular models, causing different panels of endogenous substrates phosphorylation, whose identification can be useful to understand the drug resistance phenomenon. We also compared CK2 activity of sensitive and resistant cells, and we evaluated the effect of CK2 specific inhibitors on cell viability, showing that CK2 blockade is effective in inducing cell death of resistant lines tested so far. We therefore conclude that inhibition of CK2, also considering the connections of this kinase with other survival pathways, such as Akt, can be considered as a promising tool to sensitize resistant cells to drug-induced apoptosis. c) Regulation of PRPK by Akt. Finally we analysed the functional correlation between Akt and PRPK (p53-related protein kinase), the human homologue of yeast Bud32. PRPK and Bud32 belong to a small subfamily of atypical protein kinases, called piD261. Despite Bud32, which is a kinase essential for yeast survival and morphology, the role of PRPK in the cell is still unclear. It is inactive unless it is previously incubated with cell lysates. We have seen that such an activation of PRPK is mediated by Akt, which phosphorylates PRPK at Ser250. Recombinant PRPK was shown to be phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody. Here we demonstrate that this phosphorylation takes place also in vivo: in fact cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; moreover, the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signalling pathway. The general conclusion from this work is that different survival kinases are connected and cooperate to the final purpose of ensuring a high degree of cell survival. It is therefore conceivable that even a small dis-regulation of one of them can in turn produce dramatic and pathological efforts. Thus, whenever therapeutic strategies are based on targeting one of these enzymes, this complex network of cross-talk should be always taken into account.

Proteinchinasi di sopravvivenza: CK2, Akt, PRPK. Connessioni regolatorie / Brustolon, Francesca. - (2008).

Proteinchinasi di sopravvivenza: CK2, Akt, PRPK. Connessioni regolatorie

Brustolon, Francesca
2008

Abstract

In this work I focused on functional correlations between some important protein kinases, tightly involved in cell viability. The project was divided in the following major parts: a) CK2 indirectly modulates phosphorylation state of Akt pThr308. Protein kinase CK2 is a Ser/Thr protein kinase composed of two catalytic (? and/or ?') and two regulatory (?) subunits. It is ubiquitous, constitutively active, and pleiotropic, with more than 300 protein substrates known so far. CK2 plays an antiapoptotic role, coordinating a network of signaling pathways essential for cell survival. One of these is rapresented by Akt, a kinase whose mechanism of activation is based on the phosphorylation of two key residues: Thr308 (by the PDK1 kinase) and Ser473 (by the mTOR/Rictor complex). On the other side, the dephosphorylation of Thr308 and Ser473 is accounted for by PP2A phosphatase and PHLPP, respectively. CK2 acts directly on Akt, phosphorylating its Ser129 residue, both in vitro and in vivo, thus enhancing its catalytic activity. Here we also show that downregulation of CK2 activity by inhibitors, or mutation of the CK2-dependent site in Akt (Ser129Ala), induce a lower phosphorylation of Thr308, which is not a direct target for CK2. This finding can be explained assuming that phosphorylation of Akt at Ser129 by CK2 facilitates the recognition of Thr308 by the kinase PDK1 or, alternatively, makes Akt less accessible to the phosphatase PP2A. We found that PDK1 does not discriminate between the two different forms of Akt, phosphorylated or not at the CK2 site; on the contrary, our data show that, in case of Ser129 phosphorylation, a lower degree of T308 dephosphorylation occurs. In fact, when we treat cells with the PP2A inhibitor okadaic acid, any difference at Thr308 phosphorylation is abrogated between wt Akt (phosphorylated at Ser129 by CK2) and Ser129Ala Akt mutant. We also found that Hsp90, involved in preserving the Thr308 phosphorylation state, is less tightly associated to Akt in case of the Ser129Ala mutation. This indicates that the regulation operated by CK2 on Akt can be ascribed, at least in part, to a protection from the pThr308-dephosphorylation process, possibly involving other protein partners, such as the Cdc37/Hsp90 complex. b) Drug resistance and pro-survival protein kinases. Given the pro-survival and anti-apoptotic function of CK2 and Akt, we also investigated their possible involvement in the multidrug resistance phenotype, a condition where a high proliferation rate and a reduced cell death degree result in the failure of cancer therapy. We analysed the expression level of these two kinases and their role in some cell lines, available in the two variants normally sensitive (S) or resistant (R) to chemical apoptosis. Most of the work was focused on T-lymphoblastoid cells (CEM-S and CEM-R), but we also extended our analyses to other cell models, particullary to an osteosarcoma cell line (U2OS-S and U2OS-R) and to ovarian carcinoma cells (2008-S and 2008-R). We found that CK2 and Akt are differently expressed throughout our cellular models, causing different panels of endogenous substrates phosphorylation, whose identification can be useful to understand the drug resistance phenomenon. We also compared CK2 activity of sensitive and resistant cells, and we evaluated the effect of CK2 specific inhibitors on cell viability, showing that CK2 blockade is effective in inducing cell death of resistant lines tested so far. We therefore conclude that inhibition of CK2, also considering the connections of this kinase with other survival pathways, such as Akt, can be considered as a promising tool to sensitize resistant cells to drug-induced apoptosis. c) Regulation of PRPK by Akt. Finally we analysed the functional correlation between Akt and PRPK (p53-related protein kinase), the human homologue of yeast Bud32. PRPK and Bud32 belong to a small subfamily of atypical protein kinases, called piD261. Despite Bud32, which is a kinase essential for yeast survival and morphology, the role of PRPK in the cell is still unclear. It is inactive unless it is previously incubated with cell lysates. We have seen that such an activation of PRPK is mediated by Akt, which phosphorylates PRPK at Ser250. Recombinant PRPK was shown to be phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody. Here we demonstrate that this phosphorylation takes place also in vivo: in fact cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; moreover, the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signalling pathway. The general conclusion from this work is that different survival kinases are connected and cooperate to the final purpose of ensuring a high degree of cell survival. It is therefore conceivable that even a small dis-regulation of one of them can in turn produce dramatic and pathological efforts. Thus, whenever therapeutic strategies are based on targeting one of these enzymes, this complex network of cross-talk should be always taken into account.
apoptosi, fosforilazione proteica, proteinchinasi, CK2, PKB/Akt, Akt mutante Ser129Ala, PRPK, Bud32, piD261, MDR, farmacoresistenza, cellule CEM, cellule U2OS, cellule 2008, inibitori,
Proteinchinasi di sopravvivenza: CK2, Akt, PRPK. Connessioni regolatorie / Brustolon, Francesca. - (2008).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3426386
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