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Signaling by class II PI3KC2β downstream of receptor tyrosine kinases


Borgström, Anna. Signaling by class II PI3KC2β downstream of receptor tyrosine kinases. 2013, University of Zurich, Faculty of Science.

Abstract

The family of PI3Ks (phosphatidylinositol 3-kinases) was discovered several decades ago, but until now most attention has been given to class I PI3Ks, mainly due to their previously established role in human disorders such as cancer and metabolic diseases. Class II PI3K has therefore been a bit in the shadow of the more intensively studied other families. Nevertheless, the number of reports about class II has started to increase over the past few years and we are now beginning to gain a clearer picture about the role of class II enzymes in different cellular functions and their involvement in human diseases. The fact that class II PI3K generates different second messengers (phosphoinositides) than the other PI3K family members, gives an indication that these enzymes might play a specific role in the regulation of distinct cellular functions. However, there is still a lot to be learned about the molecular mechanism of activation, the cellular function and the physiological and pathological role of class II PI3Ks. PI3KC2β belongs to class II PI3K and has a substrate specificity directed towards PtdIns and PtdIns(4)P, from which it generates PtdIns(3)P and PtdIns(3,4)P2, respectively. PI3KC2β is activated downstream of receptor tyrosine kinases (RTKs) and G protein-coupled receptors upon stimulation by growth factors such as epidermal growth factor, stem cell factor and phospholipids, including LPA (lysophosphatic acid). PI3KC2β is a monomer of 180 kDa with a prolonged C-terminus containing a C2-domain and proline rich regions in the N-terminus. The activation of class II PI3Ks is not completely defined yet, but it is known that they do not bind to any regulatory subunits. On the other hand, PI3KC2β has been shown to form multiprotein complexes leading to its activation downstream of RTKs. The complex formation between PI3KC2β and Grb2-Sos1-Abi1-Esp8 in A431 cells was shown to induce cytoskeletal rearrangements, leading to increased cell spreading and migration. Considering its important role in cell migration and cell spreading, as well as recent reports about (PI3KC2β) protein and mRNA overexpression in cancer cell lines and tumor tissue samples, a big interest in PI3KC2β and its role in tumor development and progression has developed. Nonetheless, there are many aspects of this enzyme which remain unknown and thus additional studies of PI3KC2β are encouraged. In these studies the signaling pathway modulated by class II PI3KC2β was investigated. The general approach included the targeting of PI3KC2β with isoform-specific inhibitors or transfection with small interfering RNA. Depletion of this enzyme decreased the viability of H460 and LNCap cancer cell lines and led to a decrease of S6 (ribosomal S6 protein) and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) phosphorylation levels. These changes in phosphorylation levels were independent of the activity of Akt. PI3KC2β was furthermore found to co-immunoprecipitate together with PKCβ (protein kinase C β isoform). In addition, a PKC serine substrate phosphorylation was detected in HEK293 cells transiently transfected with PI3KC2β. Interestingly, PI3KC2β phosphorylation was decreased upon treatment of the cells with Enzastaurin (PKCβ inhibitor), demonstrating the involvement of PKCβ in the serine phosphorylation of PI3KC2β. Double knock-down of PI3KC2β and PKCβ showed additive effects on cell viability and S6 phosphorylation levels. Moreover, DNA microarray analysis revealed a significant enrichment for mTOR-related genes in lung and prostate cancer samples when ranking genes according to their correlation with PIK3C2B or PRKCB expression. Finally, A431-C2β cell line overexpressing PI3KC2β was resistant to epidermal growth factor receptor inhibitors (Gefitinib and Erlotinib) and displayed a higher S6 phosphorylation level compared to the parental A431 cell line, further supporting the model that PI3KC2β regulates cell survival upstream of S6. PI3KC2β has previously been shown to become tyrosine phosphorylated upon cell stimulation with different ligands, but the impact of these phosphorylations is still unknown. In order to gain further information about the regulation of this enzyme, tyrosine site specific phospho antibodies against three newly described tyrosine residues (Y127, Y228, Y1541) were generated. In a multistep evaluation process these antibodies were shown to recognize phosphorylation and to be specific for the individual tyrosine sites. The antibodies therefore represent a useful tool for future studies of PI3KC2β phosphorylation and its impact on activity, conformation and protein interaction. All together, the results of this dissertation have contributed to the understanding of the signaling mechanism of PI3KC2β and its interaction partners. Furthermore, new tools have been generated which will be helpful in studies of activation and regulation of PI3KC2β and therefore contribute to further broaden our knowledge about this enzyme and to give a better understanding of its role in human biology and tumorigenesis.

Abstract

The family of PI3Ks (phosphatidylinositol 3-kinases) was discovered several decades ago, but until now most attention has been given to class I PI3Ks, mainly due to their previously established role in human disorders such as cancer and metabolic diseases. Class II PI3K has therefore been a bit in the shadow of the more intensively studied other families. Nevertheless, the number of reports about class II has started to increase over the past few years and we are now beginning to gain a clearer picture about the role of class II enzymes in different cellular functions and their involvement in human diseases. The fact that class II PI3K generates different second messengers (phosphoinositides) than the other PI3K family members, gives an indication that these enzymes might play a specific role in the regulation of distinct cellular functions. However, there is still a lot to be learned about the molecular mechanism of activation, the cellular function and the physiological and pathological role of class II PI3Ks. PI3KC2β belongs to class II PI3K and has a substrate specificity directed towards PtdIns and PtdIns(4)P, from which it generates PtdIns(3)P and PtdIns(3,4)P2, respectively. PI3KC2β is activated downstream of receptor tyrosine kinases (RTKs) and G protein-coupled receptors upon stimulation by growth factors such as epidermal growth factor, stem cell factor and phospholipids, including LPA (lysophosphatic acid). PI3KC2β is a monomer of 180 kDa with a prolonged C-terminus containing a C2-domain and proline rich regions in the N-terminus. The activation of class II PI3Ks is not completely defined yet, but it is known that they do not bind to any regulatory subunits. On the other hand, PI3KC2β has been shown to form multiprotein complexes leading to its activation downstream of RTKs. The complex formation between PI3KC2β and Grb2-Sos1-Abi1-Esp8 in A431 cells was shown to induce cytoskeletal rearrangements, leading to increased cell spreading and migration. Considering its important role in cell migration and cell spreading, as well as recent reports about (PI3KC2β) protein and mRNA overexpression in cancer cell lines and tumor tissue samples, a big interest in PI3KC2β and its role in tumor development and progression has developed. Nonetheless, there are many aspects of this enzyme which remain unknown and thus additional studies of PI3KC2β are encouraged. In these studies the signaling pathway modulated by class II PI3KC2β was investigated. The general approach included the targeting of PI3KC2β with isoform-specific inhibitors or transfection with small interfering RNA. Depletion of this enzyme decreased the viability of H460 and LNCap cancer cell lines and led to a decrease of S6 (ribosomal S6 protein) and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) phosphorylation levels. These changes in phosphorylation levels were independent of the activity of Akt. PI3KC2β was furthermore found to co-immunoprecipitate together with PKCβ (protein kinase C β isoform). In addition, a PKC serine substrate phosphorylation was detected in HEK293 cells transiently transfected with PI3KC2β. Interestingly, PI3KC2β phosphorylation was decreased upon treatment of the cells with Enzastaurin (PKCβ inhibitor), demonstrating the involvement of PKCβ in the serine phosphorylation of PI3KC2β. Double knock-down of PI3KC2β and PKCβ showed additive effects on cell viability and S6 phosphorylation levels. Moreover, DNA microarray analysis revealed a significant enrichment for mTOR-related genes in lung and prostate cancer samples when ranking genes according to their correlation with PIK3C2B or PRKCB expression. Finally, A431-C2β cell line overexpressing PI3KC2β was resistant to epidermal growth factor receptor inhibitors (Gefitinib and Erlotinib) and displayed a higher S6 phosphorylation level compared to the parental A431 cell line, further supporting the model that PI3KC2β regulates cell survival upstream of S6. PI3KC2β has previously been shown to become tyrosine phosphorylated upon cell stimulation with different ligands, but the impact of these phosphorylations is still unknown. In order to gain further information about the regulation of this enzyme, tyrosine site specific phospho antibodies against three newly described tyrosine residues (Y127, Y228, Y1541) were generated. In a multistep evaluation process these antibodies were shown to recognize phosphorylation and to be specific for the individual tyrosine sites. The antibodies therefore represent a useful tool for future studies of PI3KC2β phosphorylation and its impact on activity, conformation and protein interaction. All together, the results of this dissertation have contributed to the understanding of the signaling mechanism of PI3KC2β and its interaction partners. Furthermore, new tools have been generated which will be helpful in studies of activation and regulation of PI3KC2β and therefore contribute to further broaden our knowledge about this enzyme and to give a better understanding of its role in human biology and tumorigenesis.

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Additional indexing

Other titles:Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich
Item Type:Dissertation
Referees:Verrey F, Lopes Massimo, Hottiger Michael O, Arcaro Alexandre
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2013
Deposited On:07 Nov 2013 12:34
Last Modified:07 Dec 2017 23:31

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