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Regulation of the Tyrosine Kinase Itk by the Peptidyl-Prolyl Isomerase Cyclophilin A

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Regulation of the Tyrosine Kinase Itk by the Peptidyl-Prolyl Isomerase Cyclophilin A Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A Kristine N. Brazin, Robert J. Mallis, D. Bruce Fulton, and Amy H. Andreotti* Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved December 14, 2001 (received for review October 5, 2001) Interleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine ulation of the cis and trans conformers. The majority of folded kinase of the Tec family that participates in the intracellular proteins for which three-dimensional structural information has signaling events leading to T cell activation. Tec family members been gathered contain trans prolyl imide bonds. The cis con- contain the conserved SH3, SH2, and catalytic domains common to formation occurs at a frequency of Ϸ6% in folded proteins (17), many kinase families, but they are distinguished by unique se- and a small subset of proteins are conformationally heteroge- quences outside of this region. The mechanism by which Itk and neous with respect to cis͞trans isomerization (18–21). Further- related Tec kinases are regulated is not well understood. Our more, the activation energy for interconversion between cis and studies indicate that Itk catalytic activity is inhibited by the peptidyl trans proline is high (Ϸ20 kcal͞mol) leading to slow intercon- prolyl isomerase activity of cyclophilin A (CypA). NMR structural version rates (22). This barrier is a rate-limiting step in protein studies combined with mutational analysis show that a proline- folding and may serve to kinetically isolate two functionally and dependent conformational switch within the Itk SH2 domain reg- conformationally distinct molecules. These special properties, ulates substrate recognition and mediates regulatory interactions and the fact that many proline residues are highly conserved in with the active site of CypA. CypA and Itk form a stable complex homologous protein sequences, suggest that prolines may play a in Jurkat T cells that is disrupted by treatment with cyclosporin A. role in conformer-specific recognition and function. Moreover, the phosphorylation levels of Itk and a downstream Three distinct enzyme families have been identified that substrate of Itk, PLC␥1, are increased in Jurkat T cells that have catalyze the cis͞trans isomerization of prolyl imide bonds. These been treated with cyclosporin A. These findings support a novel peptidyl-prolyl cis͞trans isomerases (PPIases) (23, 24) consist of mode of tyrosine kinase regulation for a Tec family member and the cyclophilins (Cyp), FK506-binding proteins (FKBPs), and provide a molecular basis for understanding a cellular function of the parvulins (25, 26). CypA and FKBP12 are the intracellular the ubiquitous peptidyl prolyl isomerase, CypA. protein targets of the immunosuppressive natural products cyclosporin A (CsA) and FK506, respectively (27). These small ormal cell growth depends on the precise control of protein molecules inhibit PPIase activity and block T cell activation. Ntyrosine kinase activity (1). For certain families of kinases, However, the immunosuppressant activity of these small mole- the mechanism of catalytic regulation is well understood. Struc- cules derives not from inhibition of isomerase activity but rather tures of Src tyrosine kinases (2, 3) reveal intramolecular inter- from interruption of signaling events by the inhibitor–PPIase BIOCHEMISTRY actions mediated by the Src homology 2 (SH2) and Src homology complexes themselves (28). The binary CsA–CypA and FK506– 3 (SH3) domains that control catalytic activity of the neighboring FKBP12 complexes bind to and inhibit calcineurin, the serine͞ kinase domain. Specifically, distortion of the Src kinase active threonine phosphatase responsible for dephosphorylation and site is achieved in part by SH2 binding to a phosphorylated subsequent nuclear translocation of the IL-2 transcription factor, tyrosine residue in the C-terminal tail of Src (4). For other nuclear factor of activated T cells (29, 30). The PPIase inhibitors, families of kinases, the mechanistic details of catalytic regulation CsA and FK506, have shed considerable light on immunosup- remain elusive. In particular, the Tec family of nonreceptor pression as well as the signal transduction pathways involved in tyrosine kinases (5) displays distinguishing characteristics that T cell signaling, yet the normal cellular roles of the PPIases point to an alternative mode of regulation. The Tec family remain unclear. Although recent efforts have identified several kinases modulate hematopoietic cellular responses to external PPIase-mediated cellular processes (31–37), the range of normal stimuli (6). The T cell-specific Tec family member, interleukin-2 cellular functions controlled by the ubiquitous PPIases and the tyrosine kinase (Itk) (7, 8), plays a role in the maturation of molecular basis for their role in regulating signaling cascades thymocytes, is required for intracellular signaling following T have not yet been fully elucidated. cell receptor (TCR) crosslinking, and is involved in generation The cellular targets of the PPIases are likely folded proteins of second messengers that mediate cytoskeletal reorganization that contain a conformationally heterogeneous proline residue. (9). Itk is homologous to Src in the region spanning the SH3, We now present a structural analysis of Itk that places this SH2, and kinase domain but lacks the Src C-terminal tail that protein among those that exhibit multiple, well defined, low contains the regulatory tyrosine. However, activation of Itk energy conformations in solution. A single prolyl imide bond is depends on SH2-mediated interactions with phosphorylated responsible for the observed conformational heterogeneity signaling partners (9) such as Slp-76 (10) and LAT (11), sug- within Itk, and we show that this proline-dependent conforma- gesting a regulatory role for the Itk SH2 domain despite the tional switch directly regulates Itk substrate recognition. More- absence of a Src-like C-terminal regulatory tyrosine residue. over, we demonstrate that the PPIase, CypA, catalyzes the The Src regulatory mechanism highlights the role of molecular switches in controlling cellular signaling pathways. Well studied covalent modifications such as phosphorylation generate recog- This paper was submitted directly (Track II) to the PNAS office. nition sites that affect the activity and binding behavior of many Abbreviations: Itk, interleukin-2 tyrosine kinase; TCR, T cell receptor; SH2, Src homology ͞ signaling proteins (1, 12–14). Moreover, there are potential 2; SH3, Src homology 3; PPIase, peptidyl-prolyl cis trans isomerase; CypA, cyclophilin A; ͞ CsA, cyclosporin A; FKBP, FK506-binding protein; HSQC, heteronuclear single quantum regulatory switches intrinsic to polypeptides such as the cis trans correlation. isomerization of peptide bonds preceding the amino acid proline *To whom reprint requests should be addressed. E-mail: [email protected]. (15, 16). In native proteins, the local environment surrounding The publication costs of this article were defrayed in part by page charge payment. This a proline residue, as well as the overall tertiary structure, article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. influences the relative energies and therefore the relative pop- §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.042529199 PNAS ͉ February 19, 2002 ͉ vol. 99 ͉ no. 4 ͉ 1899–1904 Downloaded by guest on September 28, 2021 cis͞trans isomerization of the conformationally heterogeneous cipitated from precleared lysates with anti-Itk antibody, anti- prolyl imide bond within Itk. In vitro and in vivo functional data Zap-70 antibody (Upstate Biotechnology), or anti-PLC␥1 anti- reveal a stable CypA–Itk complex in T cells and point to a role body clone B-6-4 (Upstate Biotechnology) and protein G or for CypA in repressing Itk kinase activity. Together, these data protein A agarose. Immunoprecipitates were washed three times support a novel mode of nonreceptor tyrosine kinase regulation in RIPA lysis buffer and once in PBS resuspension buffer. and provide a molecular basis for understanding a cellular Samples were resolved on an 8% or a 12.5% (for CypA detec- function of the PPIase, CypA. tion) SDS-polyacrylamide gel. Itk detection was as described above, and Zap-70 and PLC␥1 were detected with anti-Zap-70 Materials and Methods antibody and anti-PLC␥1 antibody, respectively. CypA was Protein Expression and NMR Spectroscopy. Full-length Itk cDNA detected with anti-CypA (Calbiochem), and FKBP12 was de- (7) for PCR amplification was a gift from Leslie J. Berg tected with anti-FKBP (N-19) (Santa Cruz Biotechnology). The (University of Massachusetts Medical School). Protein sam- identity of the CypA band was confirmed by comigration with ples for NMR were expressed and purified as described purified protein purchased from Sigma. previously (38). Unless otherwise indicated, all NMR samples were 1.5 mM. NMR spectra were recorded at 25°C on a Bruker Competitive Binding Assays. Lysates and immunoprecipitated Itk DRX500 spectrometer operating at a 1H frequency of 499.867 were obtained as described above from unstimulated Jurkat T MHz. Bovine calf thymus CypA (Sigma) was used for NMR cells. The protein G agarose, containing immunoprecipitated experiments. Itk, was briefly centrifuged, and the supernatant was removed. The agarose was then resuspended
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