Structural and Biophysical Characterization of the Cytoplasmic Domains of Human BAP29 and BAP31

Two members of the B-cell associated 31 (BAP31) family are found in humans; BAP29 and BAP31. These are ubiquitously expressed receptors residing in the endoplasmic reticulum. BAP31 functions in sorting of membrane proteins and in caspase-8 mediated apoptosis, while BAP29 appears to mainly corroborate with BAP31 in sorting. The N-terminal half of these proteins is membrane-bound while the C-terminal half is cytoplasmic. The latter include the so called variant of death effector domain (vDED), which shares weak sequence homology with DED domains. Here we present two structures of BAP31 vDED determined from a single and a twinned crystal, grown at pH 8.0 and pH 4.2, respectively. These structures show that BAP31 vDED forms a dimeric parallel coiled coil with no structural similarity to DED domains. Solution studies support this conclusion and strongly suggest that an additional α-helical domain is present in the C-terminal cytoplasmic region, probably forming a second coiled coil. The thermal stability of BAP31 vDED is quite modest at neutral pH, suggesting that it may assemble in a dynamic fashion in vivo. Surprisingly, BAP29 vDED is partially unfolded at pH 7, while a coiled coil is formed at pH 4.2 in vitro. It is however likely that folding of the domain is triggered by other factors than low pH in vivo. We found no evidence for direct interaction of the cytoplasmic domains of BAP29 and BAP31.     

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt

The translocation of Akt, a serine/threonine kinase, to the plasma membrane is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon EGF stimulation of breast cancer cells, CaM co-localizes with Akt at the plasma membrane to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed NMR, biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PHD of Akt (dissociation constant = 100 nm). The interaction between CaM and Akt(PHD) is enthalpically driven, and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for binding. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P₃ binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P₃ upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.     

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (K_d) of ∼2 μm and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.     

Structural and Biophysical Characterization of the Proteins Interacting with the Herpes Simplex Virus 1 Origin of Replication

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA_15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (K_d ∼ 6 nm) followed by a second binding event (K_d ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA_15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.The initiation of DNA replication for most double-stranded DNA (dsDNA)⁶ viral genomes begins with the recognition of the origin by specific origin-binding proteins. The herpes simplex virus type 1 (HSV-1) genome encodes seven proteins required for origin-dependent DNA replication. These are the DNA polymerase (UL30) and its accessory protein (UL42), a heterotrimeric helicase-primase complex (UL5, UL8, and UL52), the single-stranded DNA-binding protein (ICP8 or UL29), and the origin-binding protein (UL9) (reviewed in Ref. 1). HSV-1 contains three functional origins, oriL and two copies of oriS. OriS, which is about 80 bp in length, consists of three UL9 recognition sites, in Boxes I, II, and III, which are arranged in two overlapping palindromes (2). Box I and Box III are part of an evolutionarily conserved palindrome that forms a stable hairpin in single-stranded DNA, which may be important in the origin rearrangement (3) during initiation of replication. Box I and II are separated by an AT-rich spacer sequence, which varies in length and nucleotide composition between the different members of the α-herpesvirus subfamily (2, 4–6).UL9 is a homodimer in solution, and EM studies, with UL9 bound to oriS, indicate the existence of a dimer or pair of dimers assembled on oriS (7). Several reports indicate that UL9 can physically interact not only with ICP8 (8) but also with other members of the HSV-1 replication complex, including UL8 (9) and UL42 (10). Thus UL9 functions as a docking protein to recruit these essential replication proteins to the viral origins. ICP8 stimulates the helicase activity of UL9 (11, 12) and binds to its C-terminal 27-aa residues (13). In the presence of ICP8, UL9 will open dsDNA containing Box I, leading to a conformational change in the origin, thus facilitating unwinding (14–16). As stated above, the changes in DNA conformation in the complete oriS may be more complex (3). Recently, it has been suggested that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by UL9. oriS* contains a hairpin formed by complementary base pairing between Box I and Box III in oriS (17). UL9, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-bp double-stranded minimal oriS fragment to oriS* and form a UL9-oriS* complex. The formation of a UL9-oriS* complex requires ATP hydrolysis (18). Therefore, the UL9-oriS* complex may serve as an assembly site for the herpesvirus replisome. Macao et al. (3) proposed a model in which full-length UL9 would be required to adopt a different conformation when binding to oriS or oriS*. The implication is that UL9 partially unwinds and introduces a hairpin into the origin of replication and that the formation of oriS* is aided, in some way, by ICP8 and requires ATP hydrolysis. Macao et al. (3) suggest that the length of the single-stranded tail of the probe DNA determines the stoichiometry of the UL9-DNA complex. oriS may bind two molecules of UL9, whereas oriS* may only bind one because the hairpin formation prevents the second interaction.Photo-cross-linking studies have shown that, although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I, suggesting that the C terminus of UL9 undergoes a conformational change upon binding to Box I (19). The results reported here are consistent with this observation. To date there is no three-dimensional structural information available on the full-length UL9 or either of the functionally characterized (helicase and DNA binding) domains. The ability to adopt different conformations and a tendency to proteolytic degradation may be responsible for this. It has been shown that UL9 binds with very high specificity to the Box I through its DNA-binding domain, consisting of the C-terminal 317 aa (UL9ct) (20, 21). Although the importance of the binding between UL9ct and oriS for the viral life cycle is well established, the mechanism behind this interaction still remains unclear. Even though UL9ct exists as a monomer in solution, uncertainty remains as to whether one or two molecules bind to a single Box I recognition sequence. Some reports have suggested that one UL9ct molecule binds to a single copy of the sequence (22–24), whereas others have proposed that UL9ct forms a dimer when bound to DNA (25, 26). This apparent difference may well result from the different protein concentrations used in different assays/experiments, which in turn highlights the difficulty of translating in vitro equilibrium experiments into cellular nonequilibrium situations.A few years ago, the crystal structure of a 60-residue C-terminal deletion mutant of ICP8 (ICP8ΔC) was determined to 3 Å resolution (Protein Data Bank code 1URJ (27)). The structure of ICP8ΔC consists of a large N-terminal domain (aa 9–1038) and a smaller entirely helical C-terminal domain (aa 1049–1120) connected to the N-terminal domain by a disordered linker (aa 1038–1049) spanning around 18 Å in the crystal structure. ICP8 preferentially binds ssDNA over dsDNA in a nonsequence-specific and cooperative manner (28). ICP8 is a zinc metalloprotein containing one zinc atom per molecule, which is coordinated by three cysteines (Cys-499, Cys-502, and Cys-510) and a histidine (His-512) (27).In this study, we show that the 60-amino acid C-terminal deletion of ICP8 (ICP8ΔC) binds strongly to UL9ct. We present three low resolution structures in solution using small angle x-ray scattering as follows: that of the UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer dsDNA (dsDNA_15-mer) containing the Box I sequence. Using these data and a variety of biophysical techniques, we demonstrate that the stoichiometries of the UL9ct-dsDNA_15-mer and UL9ct-ICP8ΔC-dsDNA_15-mer complexes are 2:1 and 2:1:1, respectively, at the micromolar protein concentrations used in this study. Using EM we visualize the assembly of the ICP8ΔC-UL9ct complex on oriS and estimate the size of the complex.     

Probing the Interactions Responsible for the Structural Stability of Trypanothione Reductase Through Computer Simulation and Biophysical Characterization

The Protein Journal - With the necessity to develop antileishmanial drugs with substrate specificity, trypanothione reductase (TryR) has gained popularity in parasitology. TryR is unique to be...     

PKC-Dependent Human Monocyte Adhesion Requires AMPK and Syk Activation

PKC plays a pivotal role in mediating monocyte adhesion; however, the underlying mechanisms of PKC-mediated cell adhesion are still unclear. In this study, we elucidated the signaling network of phorbol ester PMA-stimulated human monocyte adhesion. Our results with pharmacological inhibitors suggested the involvement of AMPK, Syk, Src and ERK in PKC-dependent adhesion of THP-1 monocytes to culture plates. Biochemical analysis further confirmed the ability of PMA to activate these kinases, as well as the involvement of AMPK-Syk-Src signaling in this event. Direct protein interaction between AMPK and Syk, which requires the kinase domain of AMPK and linker region of Syk, was observed following PMA stimulation. Notably, we identified Syk as a novel downstream target of AMPK; AICAR can induce Syk phosphorylation at Ser178 and activation of this kinase. However, activation of AMPK alone, either by stimulation with AICAR or by overexpression, is not sufficient to induce monocyte adhesion. Studies further demonstrated that PKC-mediated ERK signaling independent of AMPK activation is also involved in cell adhesion. Moreover, AMPK, Syk, Src and ERK signaling were also required for PMA to induce THP-1 cell adhesion to endothelial cells as well as to induce adhesion response of human primary monocytes. Taken together, we propose a bifurcated kinase signaling pathway involved in PMA-mediated adhesion of monocytes. PKC can activate LKB1/AMPK, leading to phosphorylation and activation of Syk, and subsequent activation of Src and FAK. In addition, PKC-dependent ERK activation induces a coordinated signal for cytoskeleton rearrangement and cell adhesion. For the first time we demonstrate Syk as a novel substrate target of AMPK, and shed new light on the role of AMPK in monocyte adhesion, in addition to its well identified functions in energy homeostasis.     

Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of Fcepsilon RI and Syk in RBL cells

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.     

Biophysical and Structural Characterization of the Thioredoxin-binding Domain of Protein Kinase ASK1 and Its Interaction with Reduced Thioredoxin

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp³¹ being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.     

Biophysical Characterization of Styryl Dye-Membrane Interactions

Styryl dyes (also referred to as FM dyes) become highly fluorescent upon binding to membranes and are often used to study synaptic vesicle recycling in neurons. To date, however, no direct comparisons of the fluorescent properties, or time-resolved (millisecond) measurements of dye-membrane binding and unbinding reactions, for all members of this family of probes have been reported. Here, we compare the fluorescence intensities of each member of the FM dye family when bound to membranes. This analysis included SGC5, a new lipophilic fluorescent dye with a unique structure. Fluorescence intensities depended on the length of the lipophilic tail of each dye, with a rank order as follows: SGC5 > FM1-84 > FM1-43 > SynaptoGreen C3 > FM2-10/FM4-64/FM5-95. Stopped-flow measurements revealed that dye hydrophobicity determined the affinity and departitioning rates for dye-membrane interactions. All of the dyes dissociated from membranes on the millisecond timescale, which is orders of magnitude faster than the overall destaining rate (timescale of seconds) of these dyes from presynaptic boutons. Departitioning kinetics were faster at higher temperatures, but were unaffected by pH or cholesterol. The data reported here aid interpretation of dye-release kinetics from single synaptic vesicles, and indicate that these probes dissociate from membranes on more rapid timescales than previously appreciated.     

Structural and Biophysical Characterization of Murine Rif1 C Terminus Reveals High Specificity for DNA Cruciform Structures

Mammalian Rif1 is a key regulator of DNA replication timing, double-stranded DNA break repair, and replication fork restart. Dissecting the molecular functions of Rif1 is essential to understand how it regulates such diverse processes. However, Rif1 is a large protein that lacks well defined functional domains and is predicted to be largely intrinsically disordered; these features have hampered recombinant expression of Rif1 and subsequent functional characterization. Here we applied ESPRIT (expression of soluble proteins by random incremental truncation), an in vitro evolution-like approach, to identify high yielding soluble fragments encompassing conserved regions I and II (CRI and CRII) at the C-terminal region of murine Rif1. NMR analysis showed CRI to be intrinsically disordered, whereas CRII is partially folded. CRII binds cruciform DNA with high selectivity and micromolar affinity and thus represents a functional DNA binding domain. Mutational analysis revealed an α-helical region of CRII to be important for cruciform DNA binding and identified critical residues. Thus, we present the first structural study of the mammalian Rif1, identifying a domain that directly links its function to DNA binding. The high specificity of Rif1 for cruciform structures is significant given the role of this key protein in regulating origin firing and DNA repair.     

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.     

Characterization of clathrin and Syk interaction upon Shiga toxin binding

Shiga toxin (Stx) is a bacterial toxin that binds to its receptor Gb3 at the plasma membrane. It is taken up by endocytosis and transported retrogradely via the Golgi apparatus to the endoplasmic reticulum. The toxin is then translocated to the cytosol where it exerts its toxic effect. We have previously shown that phosphorylation of clathrin heavy chain (CHC) is an early event following Stx binding to HeLa cells, and that this requires the activity of the tyrosine kinase Syk. Here, we have investigated this event in more detail in the B lymphoid cell line Ramos, which expresses high endogenous levels of both Syk and Gb3. We report that efficient endocytosis of Stx in Ramos cells requires Syk activity and that Syk is recruited to the uptake site of Stx. Furthermore, in response to Stx treatment, CHC and Syk were rapidly phosphorylated in a Src family kinase dependent manner at Y1477 and Y352, respectively. We show that these phosphorylated residues act as binding sites for the direct interaction between Syk and CHC. Interestingly, Syk–CHC complex formation could be induced by both Stx and B cell receptor stimulation.     

Activation Route of the Schistosoma mansoni Cathepsin B1 Drug Target: Structural Map with a Glycosaminoglycan Switch

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What Makes Ras an Efficient Molecular Switch: A Computational, Biophysical, and Structural Study of Ras-GDP Interactions with Mutants of Raf

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras^GTP) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras^GDP) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras^GDP. Most of our designed mutations narrow the gap between the affinity of Raf for Ras^GTP and Ras^GDP, producing the desired shift in binding specificity towards Ras^GDP. A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras^GDP. The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras^GDP bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras^GDP·Raf mutant complex is found in a conformation similar to that of Ras^GTP and not Ras^GDP. Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras^GTP is likely to explain the natural low affinity of Raf and other Ras effectors to Ras^GDP. Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch.     

Conformational Switch and Role of Phosphorylation in PAK Activation

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.     

Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets

Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor.