Phosphorylation as a mediator of protein function in cell migration, differentiation, and death : vimentin, AATF, and Par-4 in the spotlight
Isoniemi, Kimmo (2016-11-11)
Isoniemi, Kimmo
Åbo Akademi - Åbo Akademi University
11.11.2016
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https://urn.fi/URN:ISBN:978-952-12-3460-6
https://urn.fi/URN:ISBN:978-952-12-3460-6
Tiivistelmä
Protein phosphorylation is a major regulator of cellular signaling. It functions when a kinase joins a phosphate group to a protein, or when a phosphatase strips off a phosphate group from serine, threonine, or tyrosine residue of the protein. Protein phosphorylation is a major regulator of cellular signaling, and offers fast, dynamic, and reversible means of regulation.
Intermediate filaments (IFs) are a protein family with about 70 members, which are part of the cytoskeleton – a filamentous 3D meshwork inside the cells that engages in support, transport, and several other functions. IFs play a role in establishing and maintaining cellular organization and tissue integrity through forming cytoplasmic and nuclear filamentous structures. IFs provide structural support and assist in mechanical cellular functions. In addition, IFs work as signaling scaffolds. Through these activities, IFs regulate cellular functions such as motility, stress and apoptosis, cell growth and division, cellular differentiation, and homeostasis. Intermediate filament structure consists of conserved central rod domain and more variable N-terminal head and C-terminal tail domains. A multitude of post-translational modification (PTM) sites resides mostly in the head and tail domains. The most important of these PTMs is likely phosphorylation. Phosphorylation is known to regulate IF assembly and disassembly, and in many cases allows IFs to function as a dynamically adjustable signaling scaffold.
Vimentin is the most abundant IF, and it shows the highest expression in cells originating from mesenchyme. Vimentin is a canonical marker, and one of the causative factors of epithelial-mesenchymal transition (EMT), in which epithelial cells become more migratory and invasive, typically during development or cancerogenesis.
Our research group previously identified all the interphase in vivo serine/threonine phosphorylation sites. In our first study (I), by using phosphorylation on/off status mimicking mutagenesis, we demonstrate that from multiple sites, only phosphorylation of vimentin serines 7, 8 and/or 9 are critical to vimentin filamentous structure, and S6 and S71 and/or 72 have a lesser effect. Some phosphorylation of S7, 8 and/or 9 may be needed to stabilize or generate vimentin structure. Our results show that phosphorylation of serines 6, 7, 8 and/or 9 may affect cytoskeletal interactions, and has a major effect in increasing the tetrameric soluble pool of vimentin incrementally based on phosphorylation amount. Based on the previous research also, solubility increase is most likely caused by breakage of vimentin head-rod helix 2B interaction and warrants further investigation.
S1P and SPC are sphingolipids, which regulate processes like cellular migration, for example. In our second study (II), we demonstrate how S1P and SPC cause a restructuring of vimentin and regulate thyroid and breast cancer cell chemotactic migration via vimentin serine 71 phosphorylation. S1P receptor 2 and Rho-associated protein kinase (ROCK) appear to mediate these effects.
In the third study (III), we demonstrate how vimentin balances Notch signaling pathway by causing an increase in Jagged 1-Notch signaling at the expense of Delta-like 4-Notch signaling in angiogenesis. When vimentin is absent, Jagged 1 accumulates at the cell membrane and loses its transactivation capability. Vimentin aminoterminal PCK-regulated phosphorylation sites have a role in this. We demonstrate that this signaling effect causes weaker vasculature in the chorion allantois membrane (CAM) model, in the human umbilical vascular endothelial cell (HUVEC) angiogenesis assay, in aortic ring sprouting angiogenesis assay, and finally in vimentin knockout mouse embryos.
Par-4, CK2, AATF, and c-Jun are important regulators of cellular apoptosis and survival machinery. These proteins regulate others or are regulated by phosphorylation – CK2 being a kinase. Par-4 and CK2 function especially prominently in prostate cancer. AATF is an important component in DNA damage response.
In the fourth study (IV), we demonstrate how CK2-mediated phosphorylation of Par-4 impairs pro-apoptotic functions of Par-4. This effect involves prevention of caspase cleavage of Par-4 by Par-4 S124 phosphorylation in rats, or effects independently of cleavage by Par-4 S231 phosphorylation in humans. We also show that this phosphorylation has relevance in prostate cancer cells.
In the fifth study (V), we demonstrate previously unknown pro-apoptotic function of AATF after UV stress. We show that this mechanism requires AATF translocation from the nucleolus to nucleoplasm and interaction with transcription factor c-Jun, as well as activation of the pro-apoptotic function of c-Jun.
These studies reveal new phosphorylation-mediated mechanisms in the regulation of vimentin structure via phosphorylation of the N-terminal serine cluster, and separately via a sphingolipid-mediated pathway that also inhibits chemotactic cancer cell migration through vimentin phosphorylation. Only little studied vimentin effect on angiogenesis is described further including previously unknown effect of vimentin on Notch signaling possibly via PKC phosphorylation sites on vimentin. Our results strengthen the view of vimentin as a multifunctional cellular scaffold, and provide information about vimentin function especially in the context of cancer. Two separate studies on apoptosis describe phosphorylation-controlled CK2-Par-4-connection as potentially important in prostate cancer survival and AATF behaving pro-apoptotically after UV stress by controlling c-Jun activation by phosphorylation. Together, the results demonstrate the operational modalities of some phosphorylation-dependent switches and how these types of switches may regulate cellular fate and function in different ways.
Intermediate filaments (IFs) are a protein family with about 70 members, which are part of the cytoskeleton – a filamentous 3D meshwork inside the cells that engages in support, transport, and several other functions. IFs play a role in establishing and maintaining cellular organization and tissue integrity through forming cytoplasmic and nuclear filamentous structures. IFs provide structural support and assist in mechanical cellular functions. In addition, IFs work as signaling scaffolds. Through these activities, IFs regulate cellular functions such as motility, stress and apoptosis, cell growth and division, cellular differentiation, and homeostasis. Intermediate filament structure consists of conserved central rod domain and more variable N-terminal head and C-terminal tail domains. A multitude of post-translational modification (PTM) sites resides mostly in the head and tail domains. The most important of these PTMs is likely phosphorylation. Phosphorylation is known to regulate IF assembly and disassembly, and in many cases allows IFs to function as a dynamically adjustable signaling scaffold.
Vimentin is the most abundant IF, and it shows the highest expression in cells originating from mesenchyme. Vimentin is a canonical marker, and one of the causative factors of epithelial-mesenchymal transition (EMT), in which epithelial cells become more migratory and invasive, typically during development or cancerogenesis.
Our research group previously identified all the interphase in vivo serine/threonine phosphorylation sites. In our first study (I), by using phosphorylation on/off status mimicking mutagenesis, we demonstrate that from multiple sites, only phosphorylation of vimentin serines 7, 8 and/or 9 are critical to vimentin filamentous structure, and S6 and S71 and/or 72 have a lesser effect. Some phosphorylation of S7, 8 and/or 9 may be needed to stabilize or generate vimentin structure. Our results show that phosphorylation of serines 6, 7, 8 and/or 9 may affect cytoskeletal interactions, and has a major effect in increasing the tetrameric soluble pool of vimentin incrementally based on phosphorylation amount. Based on the previous research also, solubility increase is most likely caused by breakage of vimentin head-rod helix 2B interaction and warrants further investigation.
S1P and SPC are sphingolipids, which regulate processes like cellular migration, for example. In our second study (II), we demonstrate how S1P and SPC cause a restructuring of vimentin and regulate thyroid and breast cancer cell chemotactic migration via vimentin serine 71 phosphorylation. S1P receptor 2 and Rho-associated protein kinase (ROCK) appear to mediate these effects.
In the third study (III), we demonstrate how vimentin balances Notch signaling pathway by causing an increase in Jagged 1-Notch signaling at the expense of Delta-like 4-Notch signaling in angiogenesis. When vimentin is absent, Jagged 1 accumulates at the cell membrane and loses its transactivation capability. Vimentin aminoterminal PCK-regulated phosphorylation sites have a role in this. We demonstrate that this signaling effect causes weaker vasculature in the chorion allantois membrane (CAM) model, in the human umbilical vascular endothelial cell (HUVEC) angiogenesis assay, in aortic ring sprouting angiogenesis assay, and finally in vimentin knockout mouse embryos.
Par-4, CK2, AATF, and c-Jun are important regulators of cellular apoptosis and survival machinery. These proteins regulate others or are regulated by phosphorylation – CK2 being a kinase. Par-4 and CK2 function especially prominently in prostate cancer. AATF is an important component in DNA damage response.
In the fourth study (IV), we demonstrate how CK2-mediated phosphorylation of Par-4 impairs pro-apoptotic functions of Par-4. This effect involves prevention of caspase cleavage of Par-4 by Par-4 S124 phosphorylation in rats, or effects independently of cleavage by Par-4 S231 phosphorylation in humans. We also show that this phosphorylation has relevance in prostate cancer cells.
In the fifth study (V), we demonstrate previously unknown pro-apoptotic function of AATF after UV stress. We show that this mechanism requires AATF translocation from the nucleolus to nucleoplasm and interaction with transcription factor c-Jun, as well as activation of the pro-apoptotic function of c-Jun.
These studies reveal new phosphorylation-mediated mechanisms in the regulation of vimentin structure via phosphorylation of the N-terminal serine cluster, and separately via a sphingolipid-mediated pathway that also inhibits chemotactic cancer cell migration through vimentin phosphorylation. Only little studied vimentin effect on angiogenesis is described further including previously unknown effect of vimentin on Notch signaling possibly via PKC phosphorylation sites on vimentin. Our results strengthen the view of vimentin as a multifunctional cellular scaffold, and provide information about vimentin function especially in the context of cancer. Two separate studies on apoptosis describe phosphorylation-controlled CK2-Par-4-connection as potentially important in prostate cancer survival and AATF behaving pro-apoptotically after UV stress by controlling c-Jun activation by phosphorylation. Together, the results demonstrate the operational modalities of some phosphorylation-dependent switches and how these types of switches may regulate cellular fate and function in different ways.