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The Fyn-ADAP Axis: Cytotoxicity Versus Cytokine Production in Killer Cells.

Gerbec ZJ, Thakar MS, Malarkannan S - Front Immunol (2015)

Bottom Line: Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated.While the Fyn-PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn-ADAP pathway preferentially regulates cytokine production in NK and T cells.In this review, we discuss how the structure of Fyn controls its function in lymphocytes and the role this plays in mediating two facets of lymphocyte effector function, cytotoxicity and production of inflammatory cytokines.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, Medical College of Wisconsin , Milwaukee, WI , USA ; Department of Microbiology, Immunology and Molecular Genetics, Medical College of Wisconsin , Milwaukee, WI , USA.

ABSTRACT
Lymphocyte signaling cascades responsible for anti-tumor cytotoxicity and inflammatory cytokine production must be tightly regulated in order to control an immune response. Disruption of these cascades can cause immune suppression as seen in a tumor microenvironment, and loss of signaling integrity can lead to autoimmunity and other forms of host-tissue damage. Therefore, understanding the distinct signaling events that exclusively control specific effector functions of "killer" lymphocytes (T and NK cells) is critical for understanding disease progression and formulating successful immunotherapy. Elucidation of divergent signaling pathways involved in receptor-mediated activation has provided insights into the independent regulation of cytotoxicity and cytokine production in lymphocytes. Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated. While the Fyn-PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn-ADAP pathway preferentially regulates cytokine production in NK and T cells. In this review, we discuss how the structure of Fyn controls its function in lymphocytes and the role this plays in mediating two facets of lymphocyte effector function, cytotoxicity and production of inflammatory cytokines. This offers a model for using mechanistic and structural approaches to understand clinically relevant lymphocyte signaling.

No MeSH data available.


Related in: MedlinePlus

The domain structure and intramolecular interactions of the Fyn kinase. (A) Models of the two primary conformations of the Fyn kinase. The domain structure of Fyn consists of four SH domains. The relative positions of the domains are shown in both the inactive (left) and active (right) states. The N-terminal SH4/unique domain (blue) lies proximal to the membrane-anchoring myristoylation site. The SH4 domain is followed by the SH3 (gold) and SH2 (green) domains responsible for mediating interactions between Fyn and its target substrates. These domains are followed by a linker region that connects the SH2 domain to the bilobal SH1/kinase domain (red) responsible for enzymatic activity. The inactive form of the kinase is kept in a closed conformation by intramolecular interactions between the SH2 domain and a C-terminal phosphotyrosine as well as by an interaction between the SH3 domain and a polyproline helix in the linker region (left). The active conformation is adopted upon disruption of these interactions and opening of the kinase. In the active conformation, the SH2 and SH3 domains mediate protein–protein interactions, while the kinase domain phosphorylates downstream effectors (right). (B) Ribbon diagrams of the SH3, SH2, and SH1 domains show their relative positions in the inactive conformation. Positions are based on the structure of auto-inhibited Src (PDB deposition 2SRC), which has an identical domain arrangement. The blue high-lighted area of the SH3domain is the site of interaction with the linker region. The SH2 domain of Fyn (green) possesses an Arg residue in the second β-sheet (ArgβB5, Arg176) that is conserved across SFK family members. This Arg residue is the primary site of interaction with the phosphotyrosine on target substrates. In the inactive conformation, Arg176 is bound to the C-terminal inhibitory pTyr531 of Fyn. These intramolecular interactions place the SH2 and SH3 domains in a position to occlude the kinase domain (red) from substrate binding, thus preventing phosphate transfer. The ribbon diagram of the kinase domain is derived from PDB deposition 2DQ7, and the positions of the C-Helix and activation loop (gold) show the structure of the Fyn kinase domain complexed with staurosporine. To date, this is the only conformation of the Fyn kinase domain that has been characterized, and it is used here to demonstrate the intramolecular interactions that keep Fyn in an inactive state. The SH3 domain ribbon diagram is from PDB deposition 1NYG, and the SH2 ribbon diagram is from the PDB deposition 1AOT, and is representative of the SH2 domain bound to a phosphotyrosine-containing peptide.
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Figure 1: The domain structure and intramolecular interactions of the Fyn kinase. (A) Models of the two primary conformations of the Fyn kinase. The domain structure of Fyn consists of four SH domains. The relative positions of the domains are shown in both the inactive (left) and active (right) states. The N-terminal SH4/unique domain (blue) lies proximal to the membrane-anchoring myristoylation site. The SH4 domain is followed by the SH3 (gold) and SH2 (green) domains responsible for mediating interactions between Fyn and its target substrates. These domains are followed by a linker region that connects the SH2 domain to the bilobal SH1/kinase domain (red) responsible for enzymatic activity. The inactive form of the kinase is kept in a closed conformation by intramolecular interactions between the SH2 domain and a C-terminal phosphotyrosine as well as by an interaction between the SH3 domain and a polyproline helix in the linker region (left). The active conformation is adopted upon disruption of these interactions and opening of the kinase. In the active conformation, the SH2 and SH3 domains mediate protein–protein interactions, while the kinase domain phosphorylates downstream effectors (right). (B) Ribbon diagrams of the SH3, SH2, and SH1 domains show their relative positions in the inactive conformation. Positions are based on the structure of auto-inhibited Src (PDB deposition 2SRC), which has an identical domain arrangement. The blue high-lighted area of the SH3domain is the site of interaction with the linker region. The SH2 domain of Fyn (green) possesses an Arg residue in the second β-sheet (ArgβB5, Arg176) that is conserved across SFK family members. This Arg residue is the primary site of interaction with the phosphotyrosine on target substrates. In the inactive conformation, Arg176 is bound to the C-terminal inhibitory pTyr531 of Fyn. These intramolecular interactions place the SH2 and SH3 domains in a position to occlude the kinase domain (red) from substrate binding, thus preventing phosphate transfer. The ribbon diagram of the kinase domain is derived from PDB deposition 2DQ7, and the positions of the C-Helix and activation loop (gold) show the structure of the Fyn kinase domain complexed with staurosporine. To date, this is the only conformation of the Fyn kinase domain that has been characterized, and it is used here to demonstrate the intramolecular interactions that keep Fyn in an inactive state. The SH3 domain ribbon diagram is from PDB deposition 1NYG, and the SH2 ribbon diagram is from the PDB deposition 1AOT, and is representative of the SH2 domain bound to a phosphotyrosine-containing peptide.

Mentions: The structure of Fyn is conserved across all cell types, and in vitro characterization of the protein structure combined with in vivo characterization of Fyn function in different cell types provides a basis for understanding structure-based Fyn regulation in lymphocytes. The quaternary structure of Fyn and the consecutive arrangement of the SH4, SH3, and SH2 domains control the function of the kinase domain (SH1) through substrate recognition and regulation of enzymatic activity (Figure 1A). The N-terminal SH4 domain of Fyn is co-translationally myristoylated on the N-terminal Gly2 residue and serves as a membrane targeting and anchoring sequence (Figure 1A). The SH4 domain also contains a non-conserved region that contributes to the specific functions of SFK family members. In the case of Fyn, this unique region contains two Cys residues (Cys3 and Cys6) that can be reversibly palmitoylated. Studies using fibroblast cell lines show that palmitoylation of these residues induces localization to lipid rafts in the plasma membrane and contributes to spatial control of signaling (16, 17). The SH4 domain of Fyn is followed by consecutive SH3 and SH2 domains responsible for mediating both intermolecular and intramolecular protein interactions (Figure 1A) (18). The SH3 domain consists of a β-barrel formed by five anti-parallel β-strands (19) and two loops that form a binding pocket for linear, Pro-rich peptides. The polyproline sequences of interacting partners form helical conformations that bind with aromatic side chains of the Fyn SH3 domain. The Fyn SH2 domain is characterized by a primary binding pocket and a specificity-determining region. These two sites interact with a phosphorylated Tyr and proximal residues on binding partners (20). The SH2 and SH3 domains allow Fyn to position substrates adjacent to the bilobal tyrosine kinase (SH1) domain at its C-terminus (Figure 1A) (4). Both the N- and C-terminal lobes of the SH1 domain contain regulatory sequences (the C-Helix in the N-terminal lobe and the activation loop in the C-terminal lobe) that control ATP-binding and phosphate transfer (21–23). The kinase domain also contains a regulatory Tyr (Tyr420) that is auto-phosphorylated upon activation of enzymatic activity. Collectively, the SH domains and quaternary structure of Fyn enable it to act in multiple facets of killer cell effector function by providing the substrate specificity and control of enzymatic activity required for regulation of divergent signaling.


The Fyn-ADAP Axis: Cytotoxicity Versus Cytokine Production in Killer Cells.

Gerbec ZJ, Thakar MS, Malarkannan S - Front Immunol (2015)

The domain structure and intramolecular interactions of the Fyn kinase. (A) Models of the two primary conformations of the Fyn kinase. The domain structure of Fyn consists of four SH domains. The relative positions of the domains are shown in both the inactive (left) and active (right) states. The N-terminal SH4/unique domain (blue) lies proximal to the membrane-anchoring myristoylation site. The SH4 domain is followed by the SH3 (gold) and SH2 (green) domains responsible for mediating interactions between Fyn and its target substrates. These domains are followed by a linker region that connects the SH2 domain to the bilobal SH1/kinase domain (red) responsible for enzymatic activity. The inactive form of the kinase is kept in a closed conformation by intramolecular interactions between the SH2 domain and a C-terminal phosphotyrosine as well as by an interaction between the SH3 domain and a polyproline helix in the linker region (left). The active conformation is adopted upon disruption of these interactions and opening of the kinase. In the active conformation, the SH2 and SH3 domains mediate protein–protein interactions, while the kinase domain phosphorylates downstream effectors (right). (B) Ribbon diagrams of the SH3, SH2, and SH1 domains show their relative positions in the inactive conformation. Positions are based on the structure of auto-inhibited Src (PDB deposition 2SRC), which has an identical domain arrangement. The blue high-lighted area of the SH3domain is the site of interaction with the linker region. The SH2 domain of Fyn (green) possesses an Arg residue in the second β-sheet (ArgβB5, Arg176) that is conserved across SFK family members. This Arg residue is the primary site of interaction with the phosphotyrosine on target substrates. In the inactive conformation, Arg176 is bound to the C-terminal inhibitory pTyr531 of Fyn. These intramolecular interactions place the SH2 and SH3 domains in a position to occlude the kinase domain (red) from substrate binding, thus preventing phosphate transfer. The ribbon diagram of the kinase domain is derived from PDB deposition 2DQ7, and the positions of the C-Helix and activation loop (gold) show the structure of the Fyn kinase domain complexed with staurosporine. To date, this is the only conformation of the Fyn kinase domain that has been characterized, and it is used here to demonstrate the intramolecular interactions that keep Fyn in an inactive state. The SH3 domain ribbon diagram is from PDB deposition 1NYG, and the SH2 ribbon diagram is from the PDB deposition 1AOT, and is representative of the SH2 domain bound to a phosphotyrosine-containing peptide.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4584950&req=5

Figure 1: The domain structure and intramolecular interactions of the Fyn kinase. (A) Models of the two primary conformations of the Fyn kinase. The domain structure of Fyn consists of four SH domains. The relative positions of the domains are shown in both the inactive (left) and active (right) states. The N-terminal SH4/unique domain (blue) lies proximal to the membrane-anchoring myristoylation site. The SH4 domain is followed by the SH3 (gold) and SH2 (green) domains responsible for mediating interactions between Fyn and its target substrates. These domains are followed by a linker region that connects the SH2 domain to the bilobal SH1/kinase domain (red) responsible for enzymatic activity. The inactive form of the kinase is kept in a closed conformation by intramolecular interactions between the SH2 domain and a C-terminal phosphotyrosine as well as by an interaction between the SH3 domain and a polyproline helix in the linker region (left). The active conformation is adopted upon disruption of these interactions and opening of the kinase. In the active conformation, the SH2 and SH3 domains mediate protein–protein interactions, while the kinase domain phosphorylates downstream effectors (right). (B) Ribbon diagrams of the SH3, SH2, and SH1 domains show their relative positions in the inactive conformation. Positions are based on the structure of auto-inhibited Src (PDB deposition 2SRC), which has an identical domain arrangement. The blue high-lighted area of the SH3domain is the site of interaction with the linker region. The SH2 domain of Fyn (green) possesses an Arg residue in the second β-sheet (ArgβB5, Arg176) that is conserved across SFK family members. This Arg residue is the primary site of interaction with the phosphotyrosine on target substrates. In the inactive conformation, Arg176 is bound to the C-terminal inhibitory pTyr531 of Fyn. These intramolecular interactions place the SH2 and SH3 domains in a position to occlude the kinase domain (red) from substrate binding, thus preventing phosphate transfer. The ribbon diagram of the kinase domain is derived from PDB deposition 2DQ7, and the positions of the C-Helix and activation loop (gold) show the structure of the Fyn kinase domain complexed with staurosporine. To date, this is the only conformation of the Fyn kinase domain that has been characterized, and it is used here to demonstrate the intramolecular interactions that keep Fyn in an inactive state. The SH3 domain ribbon diagram is from PDB deposition 1NYG, and the SH2 ribbon diagram is from the PDB deposition 1AOT, and is representative of the SH2 domain bound to a phosphotyrosine-containing peptide.
Mentions: The structure of Fyn is conserved across all cell types, and in vitro characterization of the protein structure combined with in vivo characterization of Fyn function in different cell types provides a basis for understanding structure-based Fyn regulation in lymphocytes. The quaternary structure of Fyn and the consecutive arrangement of the SH4, SH3, and SH2 domains control the function of the kinase domain (SH1) through substrate recognition and regulation of enzymatic activity (Figure 1A). The N-terminal SH4 domain of Fyn is co-translationally myristoylated on the N-terminal Gly2 residue and serves as a membrane targeting and anchoring sequence (Figure 1A). The SH4 domain also contains a non-conserved region that contributes to the specific functions of SFK family members. In the case of Fyn, this unique region contains two Cys residues (Cys3 and Cys6) that can be reversibly palmitoylated. Studies using fibroblast cell lines show that palmitoylation of these residues induces localization to lipid rafts in the plasma membrane and contributes to spatial control of signaling (16, 17). The SH4 domain of Fyn is followed by consecutive SH3 and SH2 domains responsible for mediating both intermolecular and intramolecular protein interactions (Figure 1A) (18). The SH3 domain consists of a β-barrel formed by five anti-parallel β-strands (19) and two loops that form a binding pocket for linear, Pro-rich peptides. The polyproline sequences of interacting partners form helical conformations that bind with aromatic side chains of the Fyn SH3 domain. The Fyn SH2 domain is characterized by a primary binding pocket and a specificity-determining region. These two sites interact with a phosphorylated Tyr and proximal residues on binding partners (20). The SH2 and SH3 domains allow Fyn to position substrates adjacent to the bilobal tyrosine kinase (SH1) domain at its C-terminus (Figure 1A) (4). Both the N- and C-terminal lobes of the SH1 domain contain regulatory sequences (the C-Helix in the N-terminal lobe and the activation loop in the C-terminal lobe) that control ATP-binding and phosphate transfer (21–23). The kinase domain also contains a regulatory Tyr (Tyr420) that is auto-phosphorylated upon activation of enzymatic activity. Collectively, the SH domains and quaternary structure of Fyn enable it to act in multiple facets of killer cell effector function by providing the substrate specificity and control of enzymatic activity required for regulation of divergent signaling.

Bottom Line: Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated.While the Fyn-PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn-ADAP pathway preferentially regulates cytokine production in NK and T cells.In this review, we discuss how the structure of Fyn controls its function in lymphocytes and the role this plays in mediating two facets of lymphocyte effector function, cytotoxicity and production of inflammatory cytokines.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, Medical College of Wisconsin , Milwaukee, WI , USA ; Department of Microbiology, Immunology and Molecular Genetics, Medical College of Wisconsin , Milwaukee, WI , USA.

ABSTRACT
Lymphocyte signaling cascades responsible for anti-tumor cytotoxicity and inflammatory cytokine production must be tightly regulated in order to control an immune response. Disruption of these cascades can cause immune suppression as seen in a tumor microenvironment, and loss of signaling integrity can lead to autoimmunity and other forms of host-tissue damage. Therefore, understanding the distinct signaling events that exclusively control specific effector functions of "killer" lymphocytes (T and NK cells) is critical for understanding disease progression and formulating successful immunotherapy. Elucidation of divergent signaling pathways involved in receptor-mediated activation has provided insights into the independent regulation of cytotoxicity and cytokine production in lymphocytes. Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated. While the Fyn-PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn-ADAP pathway preferentially regulates cytokine production in NK and T cells. In this review, we discuss how the structure of Fyn controls its function in lymphocytes and the role this plays in mediating two facets of lymphocyte effector function, cytotoxicity and production of inflammatory cytokines. This offers a model for using mechanistic and structural approaches to understand clinically relevant lymphocyte signaling.

No MeSH data available.


Related in: MedlinePlus