Protein Tyrosine Phosphatase Receptor Type γ Is a JAK Phosphatase and Negatively Regulates Leukocyte Integrin Activation This information is current as Michela Mirenda, Lara Toffali, Alessio Montresor, Giovanni of October 1, 2021. Scardoni, Claudio Sorio and Carlo Laudanna J Immunol 2015; 194:2168-2179; Prepublished online 26 January 2015; doi: 10.4049/jimmunol.1401841 http://www.jimmunol.org/content/194/5/2168 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/01/23/jimmunol.140184 Material 1.DCSupplemental http://www.jimmunol.org/ References This article cites 46 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/194/5/2168.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Protein Tyrosine Phosphatase Receptor Type g Is a JAK Phosphatase and Negatively Regulates Leukocyte Integrin Activation Michela Mirenda,* Lara Toffali,*,† Alessio Montresor,*,† Giovanni Scardoni,† Claudio Sorio,* and Carlo Laudanna*,† Regulation of signal transduction networks depends on protein kinase and phosphatase activities. Protein tyrosine kinases of the JAK family have been shown to regulate integrin affinity modulation by chemokines and mediated homing to secondary lymphoid organs of human T lymphocytes. However, the role of protein tyrosine phosphatases in leukocyte recruitment is still elusive. In this study, we address this issue by focusing on protein tyrosine phosphatase receptor type g (PTPRG), a tyrosine phosphatase highly expressed in human primary monocytes. We developed a novel methodology to study the signaling role of receptor type tyrosine Downloaded from phosphatases and found that activated PTPRG blocks chemoattractant-induced b2 integrin activation. Specifically, triggering of LFA-1 to high-affinity state is prevented by PTPRG activation. High-throughput phosphoproteomics and computational analyses show that PTPRG activation affects the phosphorylation state of at least 31 signaling proteins. Deeper examination shows that JAKs are critically involved in integrin-mediated monocyte adhesion and that PTPRG activation leads to JAK2 dephosphoryla- tion on the critical 1007–1008 phosphotyrosine residues, implying JAK2 inhibition and thus explaining the antiadhesive role of PTPRG. Overall, the data validate a new approach to study receptor tyrosine phosphatases and show that, by targeting JAKs, PTPRG downmodulates the rapid activation of integrin affinity in human monocytes, thus emerging as a potential novel critical http://www.jimmunol.org/ regulator of leukocyte trafficking. The Journal of Immunology, 2015, 194: 2168–2179. ntegrin activation is a critical step in leukocyte recruitment regulators of integrin activation have been described, the mecha- (1). A number of protein and lipid kinases have been shown nisms regulating the on–off dynamics of kinase activity control- I to regulate the proadhesive signaling network triggered by ling integrin triggering by chemoattractants are unknown. Thus, chemoattractants (2). In this context, we have recently shown that considering the proadhesive role of JAKs, it is logical to expect protein tyrosine kinases (PTKs) of the JAK family are upstream that downmodulation of JAK activity could effect leukocyte ad- transducers linking the chemokine receptor CXCR4 to the hier- hesion dynamics. This may suggest the possibility that specific by guest on October 1, 2021 archical activation of Rho and Rap small GTPases, thus control- protein tyrosine phosphatases could be involved in the overall ling integrin affinity upregulation and homing to secondary process of chemoattractant-induced leukocyte recruitment. lymphoid organs of T lymphocytes (3). Overall, although we are The protein tyrosine phosphatases (PTP) superfamily includes still far from a complete characterization of this complex signaling receptor-like PTP (RPTP) and nontransmembrane proteins for a mechanism, it is clear that leukocyte trafficking is under tight total of 38 coding genes (4, 5). Particularly, RPTPs display a modu- control of phosphorylating events. Notably, leukocyte adhesion lar structure including a variable extracellular region, consisting is a transient phenomenon showing oscillating dynamics, cycling of different domains possibly implicated in cell–cell and cell– between adhesion and de-adhesion states propaedeutic to cell matrix adhesive contacts, and an intracellular region commonly crawling, diapedesis, and chemotaxis. However, although negative shared with other components of the superfamily. The intracel- lular region is typically composed of two domains named D1 and D2 (intracellular domain [ICD]). The catalytic activity resides in *Division of General Pathology, Department of Pathology and Diagnostics, School the D1 domain, whereas the D2 domain is possibly involved in of Medicine, University of Verona, Verona 37134, Italy; and †Center for Biomedical Computing, University of Verona, Verona 37134, Italy substrate specificity, stability, and protein–protein interaction of Received for publication July 21, 2014. Accepted for publication December 30, 2014. RPTPs. The activity of RPTPs is controlled by a variety of mech- This work was supported by Italian Association for Cancer Research Grant IG 8690, anisms (5). Although not established for all RPTPs, the main reg- by Italian Ministry of Education, Universities and Research Grant PRIN 2009, as ulatory mechanism of RPTP activity consists of the reversible well as by the Nanomedicine Project of the University of Verona and the Fondazione transition from a homodimeric inactive form to a monomeric active Cariverona. form (6, 7). Oxidative stress is also an important regulatory mech- Address correspondence and reprint requests to Prof. Carlo Laudanna, Department of Pathology and Diagnostics, Division of General Pathology, University of Verona, anism of RPTPs, leading to homodimer stabilization and hence Strada le Grazie 8, Verona 37134, Italy. E-mail address: [email protected] to the inactivation of the enzyme itself (4). The online version of this article contains supplemental material. The lack of techniques allowing specific upmodulation of PTP Abbreviations used in this article: CPP, cell-penetrating peptide; eGFP, enhanced activity in primary cells has hampered investigating the regulatory GFP; HGNC, HUGO Gene Nomenclature Committee; ICD, intracellular domain; implications of tyrosine phosphorylation/dephosphorylation turn- P1, Penetratin 1; PMN, polymorphonuclear cell; pNPP, p-nitrophenyl phosphate; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; PTPRC, PTP type over under physiological conditions. Notably, it cannot be assumed C; PTPRG, PTP receptor type g; RPTP, receptor-like PTP; TKIP, tyrosine kinase a priori whether tyrosine dephosphorylation plays, at the cellular inhibitor peptide; WD, wedge domain. level, positive or negative regulatory roles, making unclear Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 which is the best approach to study tyrosine phosphatases under www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401841 The Journal of Immunology 2169 physiological conditions. For instance, in a context where PTK ac- molecular mass, 3969.89 Da; isoelectric point, 12.02 (as calculated by tivity mediates a positive regulation, such as the role of JAKs in EMBOSS pepstats computation, http://www.ebi.ac.uk/Tools/seqstats/ integrin activation, an experimental approach based on chemical PTP- emboss_pepstats/). Peptide stock solutions (10 mM) in DMSO were kept at 280˚C and diluted in adhesion buffer immediately before the experiments. specific inhibitors or small interfering RNA silencing could be mis- All peptides were fully soluble in aqueous buffers. Standard treatment of leading. Alternatively, methods allowing selective activation of PTPs cells with peptides was for 1 h at 37˚C in 24- or 6-well plates. in primary cells are unavailable. To investigate the regulation of ty- PTPRG TAT-ICD, TAT-ICD_D1028A, and TAT–enhanced GFP (eGFP) rosine dephosphorylation in chemoattractant-triggered signal trans- fusion proteins were generated by using the pRSET-TATa expression vector, specifically designed to allow protein in-frame expression of TAT fusion duction in human leukocytes, we developed a novel approach based on proteins, as we previously described (10). Briefly, the cDNAs coding for the cell-penetrating peptides (CPPs) and allowing studying the effect of complete ICD of PTPRG (aa 797–1445) and for the complete eGFP sequence PTP receptor type g (PTPRG) activation in human primary monocyte were cloned into pRSET-TATa vector between BamHI and EcoRI sites. The ICD_D1028A cDNA was obtained by site-directed