In vitro reconstitution reveals cooperative mechanisms of adapter protein-mediated activation of phospholipase C-γ1 in T cells

Abstract

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC−γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC−γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC−γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC−γ1 activation are regulated both independently and additively by recruitment of PLC−γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC−γ1 tetramer, and by tyrosine phosphorylation of PLC−γ1. The recently solved structure of PLC−γ1 indicates that, in the resting state, several PLC−γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC−γ1, enabling contact with its membrane substrate, disinhibition of PLC−γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation. Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC−γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC−γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC−γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC−γ1 activation are regulated both independently and additively by recruitment of PLC−γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC−γ1 tetramer, and by tyrosine phosphorylation of PLC−γ1. The recently solved structure of PLC−γ1 indicates that, in the resting state, several PLC−γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC−γ1, enabling contact with its membrane substrate, disinhibition of PLC−γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation. The phospholipase C (PLC) family of enzymes contains 13 isozymes divided into six classes (β, γ, δ, ε, η, and ζ) (1Gresset A. Sondek J. Harden T.K. The phospholipase C isozymes and their regulation.Subcell. Biochem. 2012; 58: 61-94Google Scholar, 2Kadamur G. Ross E.M. Mammalian phospholipase C.Annu. Rev. Physiol. 2013; 75: 127-154Google Scholar, 3Katan M. Cockcroft S. Phospholipase C families: Common themes and versatility in physiology and pathology.Prog. Lipid Res. 2020; 80: 101065Google Scholar). These proteins are central to metazoan cellular function. Signaling pathways regulated by PLC control cellular activation, proliferation and differentiation, and mutations in these enzymes can be oncogenic or lead to autoimmunity. Recruitment and activation of PLC occur at the plasma membrane or at internal membranes where its phosphoinositide substrates are located. The very well-studied cleavage of these substrates leads to the production of diacylglycerol and inositol phosphates that in turn serve as second messengers, activate a host of enzymes directly and via the elevation of intracellular calcium (4Prole D.L. Taylor C.W. Structure and function of IP3 receptors.Cold Spring Harb. Perspect. Biol. 2019; 11a035063Google Scholar, 5Trebak M. Kinet J.-P. Calcium signalling in T cells.Nat. Rev. Immunol. 2019; 19: 154-169Google Scholar). Activation and regulation of most of the PLC isozymes are mediated by activated G proteins (6Sternweis P.C. Smrcka A.V. Regulation of phospholipase C by G proteins.Trends Biochem. Sci. 1992; 17: 502-506Google Scholar). However, the γ isoforms of the enzyme, phospholipase Cγ1 and Cγ2, differ structurally and in their mode of activation (7Hicks S.N. Jezyk M.R. Gershburg S. Seifert J.P. Harden T.K. Sondek J. General and versatile autoinhibition of PLC Isozymes.Mol. Cell. 2008; 31: 383-394Google Scholar). They contain a modular region defined by a combined nSH2-cSH2-SH3 domain that lies between a split pleckstrin homology (PH) and enzymatic domains in the enzyme. Recently, elegant structural studies demonstrated that this region lies on top of a classic phospholipase enzymatic domain, which is in a closed, autoinhibited resting state. Upon activation of the enzyme by targeted tyrosine phosphorylation of a critical regulatory residue, the authors of that study proposed that conformational changes occurred, allowing movement of the modular adapter region and leading to disinhibition of the enzyme and thus its activation (8Hajicek N. Keith N.C. Siraliev-Perez E. Temple B.R.S. Huang W. Zhang Q. Harden T.K. Sondek J. Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations.Elife. 2019; 8e51700Google Scholar). Phospholipase C-γ1 (PLC-γ1) activation has been extensively studied in the context of receptor tyrosine kinases (RTK) such as those for epidermal and fibroblast growth factors (9Beenken A. Mohammadi M. The FGF family: Biology, pathophysiology and therapy.Nat. Rev. Drug Discov. 2009; 8: 235-253Google Scholar, 10Huang Z. Marsiglia W.M. Basu Roy U. Rahimi N. Ilghari D. Wang H. Chen H. Gai W. Blais S. Neubert T.A. Mansukhani A. Traaseth N.J. Li X. Mohammadi M. Two FGF receptor kinase molecules act in concert to recruit and transphosphorylate phospholipase Cγ.Mol. Cell. 2016; 61: 98-110Google Scholar, 11Arteaga C.L. Johnson M.D. Todderud G. Coffey R.J. Carpenter G. Page D.L. Elevated content of the tyrosine kinase substrate phospholipase C-gamma 1 in primary human breast carcinomas.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10435-10439Google Scholar). Upon ligand binding to the extracellular domains of these receptors, the receptors dimerize and their cytoplasmic tyrosine kinase domains are activated by transactivation. These activated enzymatic regions trans-autophosphorylate other cytosolic tyrosines within the RTK. Specific phosphorylated tyrosines within the cytosolic domain of the RTK are then bound by signaling proteins including PLC-γ1, which contain or other protein of these molecules are phosphorylated by the RTK and critical signaling isoforms are also central to activation, in the T and is to the antigen receptor or their cytosolic protein tyrosine the enzymes are to tyrosine phosphorylated adapter In the of T cells activated by bound to a within the the membrane adapter protein for activation of T cells is a critical early substrate for the protein tyrosine kinase activated at the T cell antigen receptor W. J. J. The tyrosine kinase substrate that T cell receptor to cellular Scholar, T cell Rev. Immunol. 2009; Scholar). Specific phosphorylated tyrosine of LAT then signaling proteins and adapter molecules W. M. of and phospholipase C-γ1 with phosphorylated LAT tyrosine Biol. Scholar, The for activation of T cells signaling signaling to Biol. Scholar). These PLC-γ1, which LAT its domain, and the adapter protein Gads adapter protein of binding for the between and in T cell Biol. Scholar). In the T cell a of Gads is bound to the adapter homology domain protein of which is thus as a with Gads to LAT and regulation of signalling in and Rev. Immunol. Scholar, Q. D. Li Structural basis for binding of the domain to mode of Cell. Scholar, D. D. of in T cell activation of phospholipase C-gamma Biol. Scholar, E. J. D. S. A. M. H. A. D. the of and its substrates and Immunol. Scholar, A. E. binding of the domain to is critical for of signaling in T Cell. Biol. 2013; Scholar). bound to LAT, and also via the PLC domain and a region of SLP-76, thus a tetrameric complex D. A. of a phospholipase C-gamma 1 in for activation of 1 and Cell. Biol. Scholar). to the plasma membrane within this complex is activated by tyrosine phosphorylation of at by the T cell which is also to the D. D. and activation of the family kinase via the T cell antigen association between and Natl. Acad. Sci. U. S. A. Scholar, M. with the T cell signaling Biol. Scholar). We have previously the reconstitution of the tetrameric complex in vitro and proteins A. H. J. E. of a signaling complex upon T cell antigen receptor Natl. Acad. Sci. U. S. A. Scholar). In that study we and to define the thermodynamics of tetrameric complex formation. We that in vitro the tetrameric complex is as a of the the other formation of the complex a We that the of the complex regulation of its function in the cellular In the current study, we on the of the formation of this We in vitro with and proteins, we the complex to the of tetrameric complex formation at the of of in the plasma membrane of T These the in which is and activated at the plasma membrane of activated T We that formation of the tetrameric complex and tyrosine phosphorylation of independently the activity of in this For studies of activation of in the context of the tetrameric we protein to those we with the to A. S.N. Harden T.K. Sondek J. of activation of phospholipase Biol. Scholar). The adapter protein LAT its transmembrane domain with a at the two of the LAT intracellular tyrosines and These phosphorylated in The adapter Gads at its by of a to S. The and in protein 2012; Scholar). the and domains of the adapter for protein in vitro and of phosphorylation are in the The domain of these proteins are in The and of the proteins as in the Upon T cell activation, cytosolic is to the of the plasma membrane where with its substrate this we which are also as liposomes, of that the of the plasma and These of the in the we the substrate of PLC-γ1, and the binding to proteins in The we in to PLC enzymatic activity a by and of inositol cell or J. Scholar, Huang H. M. and inositol in Immunol. 2009; Scholar). In this the products by enzymatic cleavage of are by and by a and of the cleavage with the for binding to and thus the at of a IP3 in this demonstrated and a of to of substrate the of in this the enzyme and to the IP3 production with to We the of which has two to in the domain with this of in IP3 production at These that both the reconstitution and the enzymatic They also that of to of be for the of is that a function of the in formation of the tetrameric complex is to to the plasma membrane for contact with its substrate, binding of to the plasma we or with the other of the tetramer to and then to the of bound to the membranes as in the of the we the of recruitment of to the by to the of and binding of and of to The of to in the of the and to binding of the the content of of the or on binding of the binding of of PLC bound to with and The on and the enzymatic activity of or the and activity of the by the of the in the These that membrane recruitment of PLC-γ1, by binding of the to in the LAT is the transmembrane adapter in and the to the plasma membrane to the domain binding to LAT and the Gads domain binding LAT or We and bound to of that binding binding of to with We then bound to in this to binding to For this we with and the of previously in binding to the or with to the of to a complex binding to and binding and with the binding the complex a in enzymatic activity in the of However, and IP3 that recruitment to the for activity and by that binding to and enzymatic activity on binding to phosphorylated LAT we a of phosphorylated LAT to the and binding at and binding of to the we then that cleavage of the substrate on to and In the primary of the domain has been to be LAT N. S. H. E. of signaling is by both cooperative and Scholar). the in in vitro we a at or in to the phosphorylated LAT with the binding to the phosphorylated LAT the binding of PLC-γ1, the on activity the binding a in activity in the of which the in PLC binding to this C and in the binding or in which the LAT protein or LAT at to the However, the in activity in bound to the to the activity of bound to the phosphorylated LAT These that binding of to is the primary by which is to the membrane and The binding of the other of the tetrameric Gads and SLP-76, and the of such binding on activity then In the domain of Gads has been to with LAT or For of we have on tyrosine of LAT tyrosine to at at where binding of Gads in binding as to of of the to the tetrameric complex then 1 and for The in binding to in the of observed The of Gads SLP-76, which a binding to the and the of to Gads and also binding of of these However, the of phosphorylated to the binding of the via the Gads of and also in binding of to the bound to the membrane. the of the recruitment to In the of activity on the of Gads or and activity and by is with the that formation of the tetrameric complex at the membrane binding of Gads and in with LAT, of protein binding to in this is and phosphorylated at and The protein of is and the other proteins are The in the are the by the multiple are by the with with with PLC-γ1, with with LAT, with with with LAT, with of the is in for in a LAT in this is and phosphorylated at and The protein of is and the other proteins are The in the are the by Specific the multiple are by the with with with PLC-γ1, with with LAT, with with with LAT, with of the is in for activation of enzymatic activity phosphorylation on or tyrosine with the most studied A. S.N. Harden T.K. Sondek J. of activation of phospholipase Biol. Scholar). on this is to conformational changes in that the enzyme and thus activation of bound to a receptor tyrosine kinase (RTK) such as the epidermal growth receptor or the fibroblast growth receptor the intracellular domain of the RTK the on a also bound to that or RTK In activated T tyrosine phosphorylation of is mediated by a which is also to signaling by binding J. control kinase activation the T cell Rev. Immunol. Scholar). We a tyrosine kinase domain the to to phosphorylated molecules demonstrated activity in reconstitution Z. Marsiglia W.M. Basu Roy U. Rahimi N. Ilghari D. Wang H. Chen H. Gai W. Blais S. Neubert T.A. Mansukhani A. Traaseth N.J. Li X. Mohammadi M. Two FGF receptor kinase molecules act in concert to recruit and transphosphorylate phospholipase Cγ.Mol. Cell. 2016; 61: 98-110Google Scholar). We that in in vitro kinase the kinase domain, phosphorylated on within or molecules then in binding and enzymatic in the or of We of phosphorylation on the of to the in the or of In the enzymatic binding of to enzymatic activity as previously However, phosphorylated to demonstrated enzymatic activity to a that of in the the of and the phosphorylation of a of enzymatic However, the phosphorylation of of in a elevation in a The of the of recruitment of to the via and the phosphorylation of the enzyme on enzymatic activity of by we studied the combined of the tetrameric complex on the and the critical regulatory tyrosine on the of to the via binding the or Gads and also or phosphorylated at with in activity in the of and with the of Gads and of these the phosphorylation of at activity enzymatic activity with The by activation on these we that tetrameric complex formation and phosphorylation can independently and additively enzymatic the two have the early events that T cell receptor engagement W. J. J. The tyrosine kinase substrate that T cell receptor to cellular Scholar, T cell Rev. Immunol. 2009; Scholar, W. M. of and phospholipase C-γ1 with phosphorylated LAT tyrosine Biol. Scholar, The for activation of T cells signaling signaling to Biol. Scholar). Activation of protein tyrosine kinases at the leads in to phosphorylation of critical protein substrates such as LAT and the formation of signaling at that adapter studies and reconstitution of T cells with LAT defined the of of multiple proteins at studies to define binding and binding for the molecules to In this that tyrosine phosphorylation of LAT, the adapter molecules Gads and SLP-76, and the enzyme phospholipase C-γ1 a complex or has demonstrated that other molecules with for phospholipase C-γ1 activation also activation. These the adapter and the enzymes and with for activation of in T other proteins LAT upon activation, such as which in turn to the enzymes and with Gads for binding at phosphorylated LAT H. M. A. E. of signaling by the binding of to both LAT and Biol. Scholar, N. signaling in cells for activation of molecules to Natl. Acad. Sci. U. S. A. Scholar, M. N. N. A. H. E. at the complex are critical for J. Scholar). We have a to of the In study, we with the LAT at the central the region of the binding domain of and Gads N. S. H. E. of signaling is by both cooperative and Scholar). to define the of their the of Gads and the binding of the with via its domain, and the of a binding of with at demonstrated at demonstrated that of with the complex at a These studies thus binding and in that of the between the proteins, the of we to a tetrameric complex of be and in vitro A. H. J. E. of a signaling complex upon T cell antigen receptor Natl. Acad. Sci. U. S. A. Scholar). We a phosphorylated LAT both a for binding and a for Gads binding We the domain, which is to to and which to A. S.N. Harden T.K. Sondek J. of activation of phospholipase Biol. Scholar, H. H. and formation for and Biol. 2013; Scholar). to These with which we demonstrated that protein as a as a and proteins to at a a the and thus with the of the of tetramer formation also formation of the tetramer for the with complex formation. structure of autoinhibited recently (8Hajicek N. Keith N.C. Siraliev-Perez E. Temple B.R.S. Huang W. Zhang Q. Harden T.K. Sondek J. Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations.Elife. 2019; 8e51700Google Scholar). The study demonstrated that enzymatic domains in have structural homology with those of other In this structure of the autoinhibited enzyme, the regulatory domains including and on top of the domain in a to its to membrane studies have demonstrated that activation of phosphorylation of which is between the and domains (8Hajicek N. Keith N.C. Siraliev-Perez E. Temple B.R.S. Huang W. Zhang Q. Harden T.K. Sondek J. Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations.Elife. 2019; 8e51700Google Scholar, A. S.N. Harden T.K. Sondek J. of activation of phospholipase Biol. Scholar). The authors of the structural study that phosphorylation at this binding of to the domain and thus association of this domain with the lead to a of conformational changes and of the domains to plasma membrane the of phosphorylation of at this in study in which for structure S. S. M. M. Structural and mutations in define of for phospholipase C 2020; Scholar). enzymatic activity in T cells is mediated at the of the plasma membrane. the of tetramer formation as as the of we or the of which that of the plasma membrane. The substrate to of enzyme activity in this reconstitution Recruitment of proteins to the by and a to the protein that targeted for to the in this thus to LAT is a transmembrane protein function is to recruit enzymes including to the plasma membrane. The of LAT, as a phosphorylated also to the via the process. of LAT at a to LAT to its domain and thus recruit to the leading to enzymatic cleavage of to to the by phosphorylated LAT as as with the The recruitment of to the by activity in activation of at other phosphorylated LAT residue, in cellular studies to be of or W. M. of and phospholipase C-γ1 with phosphorylated LAT tyrosine Biol. Scholar). We the to phosphorylated this Gads via its domain as Gads and as a in the that is to this Gads the bound to phosphorylated membrane However, a of activity in the of the bound to these is as demonstrated that between and the domain A. H. J. E. of a signaling complex upon T cell antigen receptor Natl. Acad. Sci. U. S. A. Scholar, N. S. H. E. of signaling is by both cooperative and Scholar). the formation of the tetramer at the conformational changes in in its activity that be conformational changes in leading to enzymatic activation occur upon its binding to activated growth receptor tyrosine In cellular phosphorylation at its is a critical activation In this phosphorylation is mediated by a cytosolic protein tyrosine kinase of the which in T cells is J. control kinase activation the T cell Rev. Immunol. Scholar). kinase also is to the signaling complex where with and other bound proteins to to the phosphorylation this phosphorylation is likely to conformational changes leading to of enzyme domain and cleavage of the of to the tetrameric as this several other proteins and phosphorylation we phosphorylated protein in vitro with the kinase domain of the FGF phosphorylated in this binding to the However, activity to the with phosphorylated LAT, and with phosphorylated LAT, and we is a cooperative by which activation is regulated in T The formation of tetrameric complex at the plasma membrane. we have in this study, recruitment of to the membrane has on enzyme activation. However, the of the adapter proteins LAT, and within the tetrameric complex with conformational changes to activation. We have studied the of and other adapter molecules to the that are in a cooperative in that a critical tyrosine in PLC-γ1, and as in (8Hajicek N. Keith N.C. Siraliev-Perez E. Temple B.R.S. Huang W. Zhang Q. Harden T.K. Sondek J. Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations.Elife. 2019; 8e51700Google this in conformational changes in the enzyme in its activation. we have a in of this tetrameric complex by of the proteins of in this We have multiple of these regions of and that protein phosphorylation on LAT that can H. M. A. E. of signaling by the binding of to both LAT and Biol. and the transmembrane and on LAT that plasma membrane we that can to A. X. of T cell signaling Biol. Scholar). These proteins and protein domains complex that and of to the X. E. W. S. J. J. of signaling molecules T cell receptor 2016; Scholar). We that that on both and complex other and lead to a of recruitment and activation are a very as demonstrated by intracellular studies A. M. C.L. Recruitment and activation of in T into J. Scholar, J. domains and in 2019; Scholar). is also likely to be regulated and as protein complex and protein of of the complex have been observed in studies The for activation of T cells signaling signaling to Biol. Scholar). has been the of T cell activation in which a of and events occur at the of T cell activation in to of the in receptor Natl. Acad. Sci. U. S. A. Scholar, G. The for ligand and by T 2016; Scholar, A. the T cell signaling to between and Natl. Acad. Sci. U. S. A. 2020; Scholar). of the tetrameric of to and formation of the products of activity on its substrate can be of as critical steps in this complex activation in or cells and or of the and by a or with by and then by a and The for and Gads in A. H. J. E. of a signaling complex upon T cell antigen receptor Natl. Acad. Sci. U. S. A. Scholar). 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Roy Chen Li D. basis for cooperative binding of to the domain of the Scholar). with at for in The then by at for at The proteins bound to the on and by with a The of the protein by The proteins in and at and the by the in a of The for and by of and the in the on the and proteins by the Lipid 1 and then a The of and to a of for IP3 by a as in of inositol cell or J. Scholar, Huang H. M. and inositol in Immunol. 2009; Scholar). Two and to The as to to to to to to to to to to and to The the in to of IP3 to a The with with at the the as the of IP3 and on the The by and then J. In of with of a kinase domain in phosphorylation The kinase domain at and by and Z. Marsiglia W.M. Basu Roy U. Rahimi N. Ilghari D. Wang H. Chen H. Gai W. Blais S. Neubert T.A. Mansukhani A. Traaseth N.J. Li X. Mohammadi M. Two FGF receptor kinase molecules act in concert to recruit and transphosphorylate phospholipase Cγ.Mol. Cell. 2016; 61: 98-110Google Scholar). 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