Enhanced Prediction of Src Homology 2 (SH2) Domain Binding Potentials Using a Fluorescence Polarization-derived c-Met,c-Kit,ErbB, and Androgen Receptor Interactome

Many human diseases are associated with aberrant regulation of phosphoprotein signaling networks. Src homology 2 (SH2) domains represent the major class of protein domains in metazoans that interact with proteins phosphorylated on the amino acid residue tyrosine. Although current SH2 domain prediction algorithms perform well at predicting the sequences of phosphorylated peptides that are likely to result in the highest possible interaction affinity in the context of random peptide library screens, these algorithms do poorly at predicting the interaction potential of SH2 domains with physiologically derived protein sequences. We employed a high throughput interaction assay system to empirically determine the affinity between 93 human SH2 domains and phosphopeptides abstracted from several receptor tyrosine kinases and signaling proteins. The resulting interaction experiments revealed over 1000 novel peptide-protein interactions and provided a glimpse into the common and specific interaction potentials of c-Met, c-Kit, GAB1, and the human androgen receptor. We used these data to build a permutation-based logistic regression classifier that performed considerably better than existing algorithms for predicting the interaction potential of several SH2 domains.Src homology 2 protein domains (SH2)¹ are modular self-folding entities of about 100 amino acids that bind to tyrosine-phosphorylated peptide sequences contained within target proteins. The SH2 domain (1–3) was originally described nearly 20 years ago as an N-terminal region of the FES protein kinase that was not required for kinase activity but was important for its regulation. More recent studies have demonstrated that SH2 domains exist in many signaling molecules, including PLCγ1, Ras GAP, c-Src, and PI3KR. SH2 domains have been shown to enable the interaction of these signaling proteins with growth factor receptors such as FGFR1, EGFR, c-Met, and PDGFR in a phosphospecific manner (4–9). Subsequently, random peptide library screening approaches were used to define sequence motifs that resulted in the highest affinity interactions within particular SH2 domain classes (10, 11). For example, peptide sequences containing the pYEEI, pYXN, and pYMXM motifs were described to result in the highest affinity interactions with the SH2 domains from c-Src, Grb2, and the PI3KR SH2 domains, respectively. Data from such experiments have been used to generate predictions regarding the likelihood that any particular peptide sequence will interact with any particular SH2 domain (12–14).Unfortunately, the predictive performance of these algorithms has not been thoroughly empirically tested or optimized for biologically derived peptide sequences. We and others reported the first comprehensive cloning, expression, and functional analysis of human genome-encoded SH2 domains using a protein microarray-based interaction analysis approach (15–17). Similarly, peptide arrays have been used to query the interaction potential of SH2 domains with biologically derived peptide sequences in a semi-quantitative manner (18). These studies demonstrated that most biologically derived peptide sequences contained within RTKs and signaling proteins do not represent best fit sequence motifs and interact at a much lower affinity than with the optimal sequence motifs identified previously from random peptide libraries. Studies with biologically derived peptides indicated that context nonpermissive amino acids often contribute as much predictive information regarding interaction selectivity as positively contributing amino acids (19). Taken together, these results suggest that the collection of large quantitative protein interaction datasets between SH2 domains and biologically derived peptide sequences might be informative for building better algorithms that predict bona fide SH2 domain interaction sites within human protein sequences.Although protein microarrays enabled the first systems-level glimpse at SH2 domain selectivity (15, 17), they had several limitations that resulted in reduced ability to identify low affinity interactions in comparison with solution phase methods (20). We therefore designed a high throughput fluorescence polarization approach that allowed for lower affinity interactions to be defined between SH2 domains and phosphopeptides of the ErbB family of receptor tyrosine kinases (RTKs) than was possible with protein microarrays (20).RTKs are vital mediators of signal transduction in multicellular organisms. RTKs typically function as transmembrane receptors that contain a tyrosine kinase and other motifs that enable interaction with other intracellular proteins. Human cells often express many different RTK proteins from the set of 57 RTK genes encoded by the human genome (21). These RTKs may be activated in different combinations to transduce common and specific downstream signals (22). For a recent review of the complexity of RTK signaling networks, see Ref. 23. Following activation, RTKs are phosphorylated on several intracellular tyrosine residues that serve as recruitment sites for SH2 domains (15–18, 20). Activation of RTK signaling networks may cause changes in cellular motility, proliferation, survival, and cytoskeletal arrangement. Definition of their signaling capacity represents an important and unsolved problem in cell biology. Although most studies to date have focused on the role of singular RTKs in cancer progression, co-activation of RTKs derived from several unique RTK genes has recently emerged as an important driver of cancer progression (24–27). Co-activation of modules of RTKs may provide robustness against therapies designed to inhibit a single RTK (25).Herein, we profiled the interaction potential of two RTKs and two signaling proteins and compared them with the recruitment potential of the ErbB family that we have previously profiled (28). The ErbB family, c-Met, and c-Kit RTKs have been shown to drive the progression of many cancer types, including breast, head and neck, lung (29), gastrointestinal, and stomach cancers (30). Downstream adaptor proteins often augment the signaling potential of RTKs by acting as scaffolds for recruitment of many additional proteins (31–33). Therefore, we also included peptides in our study derived from the Gab1 adaptor protein, which is critical for mediating signaling networks downstream of c-Met and potentially other RTKs (34).Finally, alternative oncogenic signaling networks may have points of cross-talk with tyrosine kinase signaling networks. Steroid hormone receptors such as the androgen receptor (AR) have been shown to associate with RTKs such as EGFR (35), to be substrates of tyrosine kinases (36, 37), and to drive the progression of prostate cancer (36). We therefore queried the interaction potential of phosphopeptides derived from AR with a set of 93 of the 120 SH2 domains encoded in the human genome. We subsequently used this interaction dataset to develop a permutation-based logistic regression classifier (PEBL) for predicting the interaction potential of SH2 domains and biologically derived phosphotyrosine-containing peptides.     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP₂)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP₂-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.Membrane traffic, the transfer of material between membrane-bound compartments, is needed for such diverse cellular processes as secretion, endocytosis, and changes in cell shape that accompany cell growth, division, and migration (reviewed in references 84, 85, and 87). It is mediated by transport vesicles that are formed by budding from a donor membrane. The process of budding is driven by the assembly of a proteinaceous coat. Once the vesicle is formed, the coat must dissociate to permit fusion with an acceptor membrane and the consequent delivery of the vesicle’s contents. These steps are regulated in part by the Arf family of small GTP-binding proteins (reviewed in references 8, 23, 61, and 63). Arfs are highly conserved and are found in eukaryotes ranging from yeast to humans. The mammalian Arf family is divided into several classes based largely on sequence similarity: class I (Arfs 1 through 3), class II (Arfs 4 and 5), class III (Arf6), and the more distantly related Arf-like (Arl) class. By linking GTP binding and hydrolysis to coat assembly and disassembly, Arfs regulate membrane trafficking at a number of sites. Arf1 has been implicated in endoplasmic reticulum-to-Golgi and intra-Golgi transport, endosome-to-endosome fusion, and synaptic vesicle formation (8, 23, 28, 61, 63, 66). Arf6 has been implicated in regulation of membrane traffic between the plasma membrane and a specialized endocytic compartment, and its function has been linked to cytoskeletal reorganization (25, 26, 71, 73, 74). The specific sites of action of the other Arf family members are not known.The hydrolysis of GTP on Arf requires a GTPase-activating protein (GAP) (19, 61). With multiple Arfs and multiple sites of action, the existence of several unique Arf GAPs had been anticipated. A number of activities have been purified or partially purified from mammalian sources, including rat liver (19, 57, 77), rat spleen (21), and bovine brain (79), and two Arf GAP activities from rat liver have been resolved (77). They have similar Arf specificities but differ in their lipid dependencies. One of the Arf GAPs (ArfGAP/ArfGAP1, hereafter referred to as ArfGAP1) which functions in the Golgi is activated by dioleoglycerols (3, 4, 19, 40). ArfGAP1, in common with a yeast Arf GAP, GCS1 (72), contains a zinc finger domain which is required for activity (19). The second Arf GAP (ArfGAP2) is specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidic acid (PA). Based on lipid requirements, ArfGAP2 was speculated to function at the plasma membrane and be regulated independently of ArfGAP1 (77). ArfGAP1 and ArfGAP2 were antigenically distinct and, therefore, likely to be distinct gene products; however, prior to this study, only ArfGAP1 had been cloned (19).Src, a cytoplasmic tyrosine kinase with N-terminal Src homology 3 (SH3) and SH2 domains, transduces signals important for cell growth and cytoskeletal organization (12, 68, 91). A number of studies suggest that Src is also involved in regulating membrane traffic. Src associates primarily with endosomal membranes and in several cell types has been localized to specialized secretory vesicles, including synaptic vesicles (5, 20, 34, 46, 54, 69, 81). Overexpression of Src accelerates endocytosis (95). In addition, Src associates with or phosphorylates several proteins involved in membrane trafficking (5, 31, 43, 65).Here, we report the purification and cloning of a PIP₂-dependent Arf GAP, ASAP1. ASAP1 contains a zinc finger domain similar to that required for GAP activity in ArfGAP1 and GCS1. ASAP1 also contains a number of domains that are likely to be involved in regulation and/or localization: a pleckstrin homology (PH) domain, three ankyrin (ANK) repeats, a proline-rich region with SH3 binding motifs, and an SH3 domain. In addition, ASAP1 was identified independently as a binding protein for Src and was found to be phosphorylated on tyrosine in cells that express activated Src. ASAP1 also associated with the adapter protein c-Crk in vitro. ASAP1 was localized to the cytoplasm and the cell edge likely associated with the plasma membrane. We propose that ASAP1, by binding both Src and PIP₂, could coordinate membrane trafficking with cell growth or actin cytoskeleton remodeling.     

Context-dependent Bcl-2/Bak Interactions Regulate Lymphoid Cell Apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-x_L, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by Bcl-2 family members (1–5). This group of proteins consists of three subgroups: Bax and Bak, which oligomerize upon death stimulation to form a putative pore in the outer mitochondrial membrane, thereby allowing efflux of cytochrome c and other mitochondrial intermembrane space components; Bcl-2, Bcl-x_L, Mcl-1, and other antiapoptotic homologs, which antagonize the effects of Bax and Bak; and BH3-only proteins² such as Bim, Bid, and Puma, which are proapoptotic Bcl-2 family members that share only limited homology with the other two groups in a single 15-amino acid domain (the BH3 domain, see Ref. 6). Although it is clear that BH3-only proteins serve as molecular sensors of various stresses and, when activated, trigger apoptosis (3, 6–11), the mechanism by which they do so remains incompletely understood. One current model suggests that BH3-only proteins trigger apoptosis solely by binding and neutralizing antiapoptotic Bcl-2 family members, thereby causing them to release the activated Bax and Bak that are bound (reviewed in Refs. 9 and 10; see also Refs. 12 and 13), whereas another current model suggests that certain BH3-only proteins also directly bind to and activate Bax (reviewed in Ref. 3; see also Refs. 14–17). Whichever model turns out to be correct, both models agree that certain antiapoptotic Bcl-2 family members can inhibit apoptosis, at least in part, by binding and neutralizing activated Bax and Bak before they permeabilize the outer mitochondrial membrane (13, 18, 19).Much of the information about the interactions between pro- and antiapoptotic Bcl-2 family members has been derived from the study of synthetic peptides corresponding to BH3 domains. In particular, these synthetic peptides have been utilized as surrogates for the full-length proapoptotic proteins during structure determinations (20–22) as well as in functional studies exploring the effect of purified BH3 domains on isolated mitochondria (14, 23) and on Bax-mediated permeabilization of lipid vesicles (15).Recent studies using these same peptides have suggested that interactions of the BH3 domains of Bax, Bak, and the BH3-only proteins with the “BH3 receptors” of the antiapoptotic Bcl-2 family members are not all equivalent. Surface plasmon resonance, a technique that is widely used to examine the interactions of biomolecules under cell-free conditions (24–26), has demonstrated that synthetic BH3 peptides of some BH3-only family members show striking preferences, with the Bad BH3 peptide binding to Bcl-2 and Bcl-x_L but not Mcl-1, and the Noxa BH3 peptide binding to Mcl-1 but not Bcl-2 or Bcl-x_L (27). Likewise, the Bak BH3 peptide exhibits selectivity, with high affinity for Bcl-x_L and Mcl-1 but not Bcl-2 (12). The latter results have led to a model in which Bcl-x_L and Mcl-1 restrain Bak and inhibit Bak-dependent apoptosis, whereas Bcl-2 does not (10).Because the Bak protein contains multiple recognizable domains in addition to its BH3 motif (28, 29), we compared the binding of Bak BH3 peptide and Bak protein to Bcl-2. Surface plasmon resonance demonstrated that Bcl-2 binds Bak protein with much higher affinity than the Bak 26-mer BH3 peptide. Further experiments demonstrated that the K_D for Bak differs among naturally occurring Bcl-2 sequence variants but is only 3-fold higher than that of Mcl-1 in some cases. In light of previous reports that Bcl-2 overexpression contributes to neoplastic transformation (30–33) and drug resistance (34–36) in lymphoid cells, we also examined Bcl-2 expression and Bak binding in a panel of neoplastic lymphoid cell lines. Results of these experiments demonstrated that Bcl-2 expression varies among different lymphoid cell lines but is up to 40-fold more abundant than Mcl-1. In lymphoid cell lines with abundant Bcl-2, Bak is detected in Bcl-2 as well as Mcl-1 immunoprecipitates; and Bak-dependent apoptosis induced by Mcl-1 down-regulation can be prevented by Bcl-2 overexpression. Collectively, these observations shed new light on the role of Bcl-2 in binding and neutralizing Bak.     

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu⁵⁴³-Asp⁵⁴⁴ in this linker or Leu⁶¹¹, Arg⁶⁸³, or Phe⁶⁹⁴ in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.The Janus family of tyrosine kinases (JAKs)² are key regulators of cytokine receptor signaling in hematopoiesis and immune responses (1). Of the four mammalian JAK kinases, JAK2 transmits signals for a variety of cytokine receptors, including the erythropoietin receptor (EpoR) that is essential for red blood cell production (2). Upon Epo stimulation, JAK2 activates downstream signaling, such as STAT5, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/AKT pathways (2). Mice deficient in Epo, EpoR, or JAK2 die embryonically due to the absence of definitive erythropoiesis (3–5).In addition to regulation by phosphatases and suppressors of cytokine signaling (6, 7), JAK2 kinase activity is critically controlled by an autoinhibitory mechanism. Like other JAK members, JAK2 contains an N-terminal segment followed by a pseudokinase domain and a C-terminal tyrosine kinase domain. The N-terminal segment, consisting of a FERM (protein 4.1, ezrin, moezin, radixin homologous) domain and an atypical SH2 domain (1), mediates association with the membrane-proximal region of the cytokine receptors (8). Binding of JAK2 through its N-terminal segment to the EpoR is essential for EpoR surface expression (9). The pseudokinase domain is predicted to adopt a kinase fold but lacks residues essential for catalysis (10). Deletion of the pseudokinase domain leads to a marked increase in JAK2 kinase activity and loss of response to cytokine stimulation (11–13). Therefore, this domain is essential for JAK2 autoinhibition and is essential for JAK2 activation upon cytokine stimulation. Consistent with this notion, a point mutation in the JAK2 pseudokinase domain was identified in the majority of myeloproliferative disorder patients, including 90% of polycythemia vera (PV) patients (14–18). This mutation, V617F, in the presence of a dimerized receptor scaffold, such as the EpoR, resulted in the constitutive activation of JAK2 and downstream signaling effectors (19, 20) and caused erythrocytosis in a murine bone marrow transplant model (14, 21–23). Recently, mutations immediately adjacent to the JAK2 pseudokinase domain in the SH2-pseudokinase domain linker were identified in PV patients and shown to cause constitutive activation of JAK2 and a PV-like phenotype in mice (24–26). The molecular mechanisms underlying the control of JAK2 activity (i.e. the swift augmentation of its activity upon receptor activation) are poorly understood. The residues involved in the autoinhibition in JAK2 are unknown.In this work, we sought to characterize the regulatory mechanisms controlling JAK2 kinase activity. Using a functional screen for activating JAK2 mutations that signal constitutively, we discovered 13 mutations in the pseudokinase domain and in the SH2-pseudokinase domain linker. These mutations identified specific residues that are important for the inhibition of basal JAK2 kinase activity and for cytokine-induced JAK2 activation. In addition, we showed that the SH2-pseudokinase domain linker is essential for interaction with the EpoR, autoinhibitory regulation, and Epo-inducible JAK2 activation and may act as a switch in relaying cytokine receptor engagement to JAK2 activation by flexing the pseudokinase domain hinge.     

Ubiquitin-mediated Degradation of the Formin mDia2 upon Completion of Cell Division

Formins assemble non-branched actin filaments and modulate microtubule dynamics during cell migration and cell division. At the end of mitosis formins contribute to the generation of actin filaments that form the contractile ring. Rho small GTP-binding proteins activate mammalian diaphanous-related (mDia) formins by directly binding and disrupting an intramolecular autoinhibitory mechanism. Although the Rho-regulated activation mechanism is well characterized, little is known about how formins are switched off. Here we reveal a novel mechanism of formin regulation during cytokinesis based on the following observations; 1) mDia2 is degraded at the end of mitosis, 2) mDia2 is targeted for disposal by post-translational ubiquitin modification, 3) forced expression of activated mDia2 yields binucleate cells due to failed cytokinesis, and 4) the cytokinesis block is dependent upon mDia2-mediated actin assembly as versions of mDia2 incapable of nucleating actin but that still stabilize microtubules have no effect on cytokinesis. We propose that the tight control of mDia2 expression and ubiquitin-mediated degradation is essential for the completion of cell division. Because of the many roles for formins in cell morphology, we discuss the relevance of mDia protein turnover in other processes where ubiquitin-mediated proteolysis is an essential component.Formin proteins play a role in diverse processes such as cell migration (1, 2), vesicle trafficking (3, 4), tumor suppression (5, 6), and microtubule stabilization (7, 8). Formins also play an essential and conserved role in cytokinesis (9–11). Proper cell division is essential in all animals to maintain the integrity of their genome. Failure to complete cytokinesis can result in genomic instability and ultimately lead to disease such as cancer (12).The members of the mDia² family of formins are autoregulated Rho effectors that remodel the cytoskeleton by nucleating and elongating non-branched actin filaments (13). The amino terminus of mDia contains a GTPase binding domain (GBD) that directs interaction with specific Rho small GTP-binding proteins. The adjacent Dia inhibitory domain (DID) mediates mDia autoregulation through its interaction with the carboxyl-terminal diaphanous autoregulatory domain (DAD) (14, 15). Between the DID and DAD domains lie the conserved formin homology 1 (FH1) and FH2 domains. The FH1 domain is a proline-rich region that mediates binding to other proteins such as profilin, Src, and Dia-interacting protein (16–19). In contrast, the FH2 domain binds monomeric actin to generate filamentous actin (F-actin) and can also bind microtubules directly to induce their stabilization (8, 20).Although the mechanism of mDia activation is well characterized, little is known about its inactivation. Previous reports have suggested that formins can cycle between active, partially active, and inactive states (21, 22) due to GTP hydrolysis upon Rho binding to GTPase-activating proteins. Another formin inactivation mechanism is through mDia interactions with Dia-interacting protein (23). In the context of cortical actin assembly, Dia-interacting protein negatively regulates mDia2 actin polymerization but has no effect on mDia1 actin polymerization despite its ability to interact with both proteins directly (17). Because of the fundamental role for formins in cell division, we sought to identify how mDia2 is inactivated in mitosis.During cell division, the expression level and activity of many proteins (e.g. cyclins and Aurora and Polo kinases) are tightly regulated (24). A unifying regulatory mechanism among these proteins is ubiquitin-mediated proteolysis. In this study we find that mDia2 protein levels are constant from S phase into mitosis and dramatically decrease at the end of mitosis due to ubiquitin-mediated degradation. Failure to inhibit mDia2 actin assembly results in multinucleation, which supports an essential role for the tight regulation of mDia2 during cell division.     

The Development and Application of a Quantitative Peptide Microarray Based Approach to Protein Interaction Domain Specificity Space

Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology.Protein interaction domains (PIDs)¹ often compete for the same linear motif binding sites across a range of affinities, resulting in many potential interactions that may enable the rapid assembly and disassembly of signaling proteins in response to external and internal cues (1, 2). PID-peptide interactions have small binding interfaces, resulting in moderate affinity interactions mediated primarily by a few amino acid “hot-spots” within motifs specific for a particular PID family (3–5). The power of individual residues to direct interactions, the absence of structural constraint for linear motifs, and the modularity of PIDs has enabled the rapid evolution of these networks resulting in many large multimember PID families in higher eukaryotes (6–9). For these large families dedicated to the recognition of similar ligands, PID specificity—or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs—underpins the effective conveyance of specific cell signals. High throughput interaction mapping efforts are used to decipher how this PID “specificity space” is populated, thereby providing insight into protein function and the principles of network architecture and evolution (10–15). The extent of binding overlap or interaction promiscuity within and between PID families for physiological ligands, the affinity range of overlapping interactions, and the biological relevancy of these interactions are important questions thus far poorly resolved by existing high throughput methods. Here, we develop and apply a quantitative high throughput method capable of addressing these questions.Peptide arrays (16, 17), degenerate libraries (18, 19), and phage display (20) are the most frequently applied high throughput approaches for investigating PID specificity. Phage display and degenerate library approaches sample a large ligand space and can produce consensus selectivity motifs that represent the most preferred residues at every position panned. This selectivity data is used to predict interactions, often via position specific scoring matrices (PSSMs) (21–23). However, neither approach can explicitly measure nonbinding events and only large phage display data sets can resolve a limited subset of high-affinity contextual binding information (24). Nonbinding information and contextual interplay, that is, correlated contributions between ligand positions to binding affinity, play important roles in defining the specificity landscapes for multiple PID families (25–27). Having explicit nonbinding or low-affinity information available helps uncover contextual binding information, and improves the accuracy of interaction priority assignment between multiple competing PIDs. Correspondingly, the availability of nonbinding and contextual information improves interaction prediction performance (28, 29). Peptide arrays using physiological ligands do not have these limitations, yet may under-sample PID specificity space because of smaller library sizes. Newly emerging ultrahigh density peptide arrays avoid this particular limitation and are capable of sampling the entire proteome (30). However, a common limitation for all of these techniques is their dependence on nonquantitative interaction information.A comprehensive understanding of PID specificity space requires the quantitative assessment of pairwise interactions across a broad dynamic range of affinities. Common low-throughput biophysical techniques used to measure protein-peptide interaction affinities require highly pure and often large amounts of interactants along with prior knowledge of their interaction. To facilitate discovery and lessen the stringency of the purity and/or quantity requirements of interacting molecules, multiple quantitative high throughput methodologies have been developed. Thus far, the protein microarray (PMA) (31) and high-throughput fluorescence polarization (HTFP) (11, 32) quantitative approaches have been used to examine PID specificity. Unfortunately, PMAs suffer from poor sensitivity, reproducibility, and measurement discrepancies (32, 33). The alternative HTFP assay is more sensitive than PMAs, but also has poor reproducibility and is biased toward high affinity interactions (32). Further, PMAs and HTFP have minimal K_D sensitivities of 2 and 20 μm, respectively. This boundary limits their scope of application considering the importance of moderate affinity interactions for many PID families (34) and the general importance of these interactions in directing emergent phenomena such as ultrasensitivity and interaction gating (35, 36).The Src Homology 2 (SH2) domain phosphotyrosine interactome is a system of prominent physiological importance that has been the subject of multiple preceding interaction mapping and platform development efforts (17, 19, 31, 32, 37, 38). Tyrosine phosphorylation and its recognition by SH2 domains is a uniquely metazoan adaptation (39), prominently involved in the transduction of growth signals (1, 2, 40). SH2 domain specificity has been leveraged in multiple contexts to rewire signaling pathways (15), and specificity dysregulation is associated with multiple diseases, including cancer (40–42). Despite the importance of directing specific signals, many SH2 domains share sequence preferences, resulting in substantial binding crossover over a broad dynamic range of affinities (27). Yet, the extent of this crossover and the peptide motif characteristics responsible for its direction are poorly resolved. In light of these biological attributes and the availability of preceding information to guide platform development and assess its performance, we chose a significantly novel set of 124 physiological phosphopeptides and four SH2 domains as a pilot interactome for our arrays.Here we develop a cellulose peptide conjugate microarray (CPCMA) based approach that is the first broadly applicable protein interaction mapping approach capable of explicitly capturing binding and nonbinding information, resolving the contextual interplay between amino acids that contribute to binding, and providing a quantitative measure of interaction affinity. We optimize multiple production and incubation parameters for the arrays and develop an experimental and computational pipeline to control for systematic artifacts and robustly estimate affinities, ultimately producing a physiological interactome of 368 measurements between 92 peptides and four domains. In contrast to preceding platforms, we find our approach is highly sensitive, extremely precise, and consistent with multiple forms of orthogonal biophysical and in vivo validation. Leveraging unique modes of analysis enabled by the CPCMAs, we demonstrate that SH2 domains vary considerably in promiscuity and binding dynamic range in a manner aligned with biological function. Consistent with recent evidence that pathway crosstalk is subjected to negative selection (10–12, 43) we demonstrate for the first time that the degree distribution of the interactome is a function of affinity, and therefore little binding overlap is found among the highest affinity interactions, suggesting that such sites may be important points of in vivo coregulation. We highlight a few such interactions and corroborate others using available domain-site specific in vivo interaction information. Next, in order to provide insight into the specificity determinants responsible for this segregation we produce for the first time predictive domain specific binding motif affinity matrices. Lastly, we demonstrate the unique ability of the CPCMAs to resolve contextual contributions to binding, and highlight examples where contextual information contributes to binding specificity.Quantitative analysis of systems-level PID properties is a necessary prerequisite for improved mathematical modeling of signal transduction systems, specificity focused drug design, and engineering advanced synthetic biology circuits. Thus, our quantitative CPCMA based approach should have broad application in future network biology and PID specificity efforts.     

A Comprehensive Immunoreceptor Phosphotyrosine-based Signaling Network Revealed by Reciprocal Protein–Peptide Array Screening

Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM), or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein–protein interactions in a systematic and reliable manner.Immune receptor signaling, critical for proper immune response, involves signaling pathways mediated by specific tyrosine residues that are phosphorylated upon receptor activation (1). These tyrosine phosphorylation sites are frequently found in one of the three types of immunoreceptor tyrosine-based regulatory motifs (ITRMs)¹. The immunoreceptor tyrosine-based activation motifs (ITAMs) with the consensus sequence YxxI/Lx(6–12)YxxI/L, where x represents any amino acid, are typically associated with positive or activating immune response (2). In contrast, the immune system elicits negative or inhibitory response through receptors bearing the immunoreceptor tyrosine-based inhibition motifs (ITIMs) with the degenerated sequence S/I/V/LxYxxI/V/L (3). Interestingly, SLAM/CD150 and related receptors of the CD2 subfamily contain a third type of signaling motif called Immunoreceptor Tyrosine-based Switch Motif (ITSM) with the consensus sequence TxYxx(V/I) (4). An ITSM can convey either an activating or inhibitory signal, depending on the type of immune cell, the receptor and the bound protein (5).In general, phosphorylated ITIMs recruit the SH2 domain-containing tyrosine phosphatase SHP-1 or SHP-2 (6), and phosphorylated ITAMs are recognized by protein tyrosine kinases such as ZAP70 in T cells and SYK in B cells (7). Nevertheless, these distinctions are only relative and both motifs may be involved in either positive or negative immune functions. For example, ITIM-containing inhibitory receptors found on phagocytes can suppress or enhance inflammatory cytokine production depending on the downstream proteins that they recruit (6). Similarly, ITAM sequences have been found to mediate inhibitory signaling (8). Indeed, even within the same cell type, an ITAM-containing receptor may mediate functions as diverse as microbial killing, antigen presentation, cytokine production, T-Cell instruction, and tissue repair (2). Moreover, exquisite binding specificities have been observed for different ITRMs. For example, the CD31 ITIMs recruit SHIP-1, SHP-1, and SHP-2 (9). In contrast, the archetypal ITIM identified in the cytoplasmic domain of the inhibitory IgG Fc receptor FcγRIIB (10) recruits SHIP-1 and -2, but not SHP-1 or -2. Although the FcγRIIB ITIM has an affinity for the SH2 domain of either SHP-1 or -2, it does not recruit these tyrosine phosphatases as do other ITIM-bearing receptors (11). Besides SHIP-1/2 and SHP-1/2, other SH2-containing proteins have also been found associated with certain ITIMs. For example, through their respective ITIM sequences, the LAIR-1 receptor recruits tyrosine kinase CSK (12), Lax interacts with GRB2, PIK3R1 and GRAP2 (13), and CD72 forms a complex with SHP-1 and GRB2 (14). These findings indicate that immunoreceptor signaling is context-dependent and that the precise pairing of an ITRM sequence with an SH2-containing protein plays a critical role in dictating the signaling and biological outcome.Given the critical importance of SH2 domain-phosphotyrosine (pY) interaction in immune signaling, we set to systematically identify SH2 domain-ITRM interactions. A variety of different high-throughput methods have been developed for the identification of protein–protein or protein-peptide interactions to date. These include yeast two-hybrid (15), affinity purification coupled to mass spectrometry (AP-MS) (16), phage display (17), and protein (18) and peptide arrays (19). We employed protein and peptide arrays in our study because of their simplicity and amenability to post-translational modifications. Specifically, we combined SH2 domain array with phosphotyrosine peptide array to decipher an SH2-ITRM interactome for immunoreceptors. We showed that the peptide and SH2 domain arrays were not simple mirror images of each other, but rather, they exhibited distinct, but complementary binding patterns. Although neither array platform alone was sufficient to detect all authentic interactions, integration of data from reciprocal peptide and protein array screens allowed us to identify a high-confidence SH2-ITRM interactome. The interactome has not only provided a systematic view of immunoreceptor signaling network, but also generated unique insights into the signaling specificity for the different immune signaling motifs.     

In Vivo Mutational Analysis of YtvA from Bacillus subtilis: MECHANISM OF LIGHT ACTIVATION OF THE GENERAL STRESS RESPONSE

The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates σ^B through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light, oxygen, and voltage) domain and a C-terminal STAS (sulfate transporter and anti-sigma factor) domain and shows light-modulated GTP binding in vitro. Here, we examine the mechanism of YtvA-mediated activation of σ^B in vivo with site-directed mutagenesis. Constitutive off and constitutive on mutations have been identified. Disruption of GTP binding in the STAS domain eliminates light activation of σ^B. In contrast, modification of sites relevant for phosphorylation of STAS domains does not affect the stress response significantly. The data obtained are integrated into a model for the structure of full-length YtvA, which presumably functions as a dimer.LOV² domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted Jα (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8, 9). Stress via the addition of salt or ethanol (for a review, see Ref. 10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13, 14). YtvA, which mediates light activation of σ^B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16).When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9, 14, 17, 18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating σ^B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of σ^B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180° upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central β-sheet and the C-terminal linker region, the Jα-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the σ^B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavin-binding pocket, the β-sheet of the LOV domain, and the GTP-binding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approach was used to model the structure of full-length YtvA. The model suggests that light modulates accessibility of the GTP-binding site of the STAS domain of YtvA.     

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)¹. Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3^N and microscale thermophoresis (MST) indicates an intermediate binding affinity (K_d = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.T cell migration and the establishment of productive T cell/APC interactions are regulated by the activity of integrins. In resting T cells, integrins are expressed in an inactive state that adopts a conformation with low affinity for their ligands. Members of the intercellular adhesion molecule family (ICAM 1–5) are the physiological ligands of lymphocyte function-associated antigen 1 (LFA-1, αLβ2-integrin) whereas vascular cell adhesion molecule (VCAM) and fibronectin are the ligands for the β1-integrin very late antigen 4 (VLA-4) (1, 2). Triggering of the T cell receptor (TCR) by peptide-major histocompatibility complex (MHC) or stimulation of chemokine receptors (e.g. CCR7 with CCL21 or CXCR4 with CXCL12) induces a conformational change of the integrins that increases their ligand binding (affinity regulation) and subsequently mediates clustering of integrins at the cell surface (avidity regulation). The intracellular events leading to integrin activation have collectively been termed inside-out signaling. Conversely, ligand-bound integrins transmit a signal to the T cell and thereby promote adhesion, activation, proliferation, and migration of T cells (outside-in signaling) (1, 2).In both inside-out and outside-in signaling pathways tyrosine phosphorylation of adaptor proteins, either present as transmembrane scaffolds or as transiently membrane-anchored proteins, is a crucial primary event in signal transmission to integrins.An essential functional module operating at the integrin-membrane-cytoskeleton interface contains the cytosolic adaptor protein ADAP at its core. Ablation of ADAP in mice leads to dysfunctional integrin clustering and activity, thus compromising the adhesive and migratory properties of these cells. In addition to its instantaneous effects on cellular motility, ADAP was shown to act as a regulator of NFκB p65 nuclear translocation (3), a function that might well contribute to the observed modulation of cytokine production, like interleukin-2 (4, 5). A contribution of ADAP to mast cell degranulation has been postulated (6), and its complex formation with cytoskeletal regulators during early phases of phagocytosis in macrophages has been recognized early on (7). ADAP is also critical for normal platelet adhesion (8) and mutations in the human protein have recently been suggested to form an underlying genetic cause for autosomal recessive thrombocytopenia (9).ADAP interacts with several effectors of T cell function, either constitutively or phosphorylation-dependent. The SH3 domain of SKAP55 (Src-kinase associated phosphoprotein of 55 kDa) interacts with a proline-rich sequence (PRS) stretch in ADAP (Fig. 1A) (10, 11), whereas another PRS (FPPPP) is responsible for the interaction with the actin regulator Ena/VASP-like protein (EVL) (12). Membrane binding of the ADAP-SKAP55 complex is conferred by the PH domain of SKAP55 and to a lower extend by the C-terminal hSH3 domain (hSH3^C) of ADAP (13–16). Moreover, ADAP is strongly tyrosine-phosphorylated upon TCR stimulation and thereby serves as a hub for SH2 domain-containing proteins such as SLP76, FYN and NCK (Fig. 1A) (17–21). Beside these well characterized interactions, several other SH2-domain containing binders were identified by pull-down approaches using phosphorylated peptide baits (Fig. 1A) (22, 23). Most of the so far characterized SH2-pTyr interactions in ADAP are mapped to unstructured regions. An exception is Y571, which is located in close proximity to the folded hSH3^N domain of ADAP. Phosphoproteomic profiling of activated T cells has identified Y571 as a major phosphorylation site in ADAP (18, 24–30). Our rationale for the experimental approach chosen here was that the constraints imposed by the folding of the hSH3 domains impact the choice of SH2 domains that bind to such motifs in ADAP.Open in a separate windowFig. 1.Phosphotyrosine sites of ADAP and interaction partners. A, Schematic overview of the ADAP primary structure indicating interaction partners of different phosphotyrosine sites identified by peptide pull-down approaches (22, 23). Black arrows show SH2-pTyr interaction sites. White arrows show interactions dependent on proline rich sequences (PRS). B, Fyn kinase catalyzed in vitro phosphorylation of full-length ADAP. Identification and relative quantification of phosphorylation degrees of individual tyrosine residues was obtained by mass spectrometry. Phosphorylation degrees were estimated by comparing relative MS peak intensities of the corresponding peptide/phosphopeptide pairs as described (33, 34).To meet this challenge, we employed interaction proteomics and immunoprecipitation experiments using different constructs of ADAP that comprise the major phosphotyrosine sites including the two folded hSH3 domains. In particular, Y571, a residue residing at the domain border of the hSH3^N domain, is shown to interact with proteins that are not identified by the peptide pull-down approach. We identify the ZAP70 kinase as the most robust binding partner of pY571 and show that this interaction is maintained in primary T cells. Using NMR spectroscopy we further show that the N-terminal SH2 domain of ZAP70 is responsible for binding and we confirm the direct interaction between the two proteins to be inducible upon T cell receptor or CXCR4 stimulation. Abolishing the interaction by using a Y571F mutation compromises T cell migration along a CXCL12 gradient but does not affect TCR- and CXCR4-mediated adhesion and T cell interaction with APCs. In this way, the Y571F mutant of ADAP provides the striking example of a molecular switch that selectively invokes the migratory, but not the adhesive, signaling pathways in T cells.