Nishizuka and colleagues discovered protein kinase C (PKC) as a calcium-dependent, lipid cofactor-sensitive protein kinase . InitialBox 1PKC serve as a paradigm for the reversible regulation of membrane localization by the concerted action of two membrane-targeting modules.C1: a cysteine-rich region of approximately 50 residues present in all PKC isozymes. In cPKCs and nPKCs, it is present as a tandem repeat C1A and C1B. aPKCs contain a single copy of the domain termed atypical because it does not bind phorbol esters. The domain contains two pulled apart β-sheet forming the ligand-binding pocket. Two zinc atoms are coordinated by two histidines and six cysteines at opposite ends of the primary sequence stabilizing the domain. In aPKCs, one face of the ligand-binding pocket is compromised, so that the module cannot bind phorbol esters or DAG.Ligand binding dramatically alters the surface properties of the module (C1B). The ligand caps the hydrophilic ligand-binding pocket, so that the top-third of the C1 domain presents a continuous hydrophobic surface thus achieving membrane targeting by simply altering membrane properties.C2: an independent membrane-targeting module that binds calcium in cPKCs, but not in nPKCs. C2 domain is a β-strand rich globular domain with loops formed by sequences at opposite ends of the primary structure. Two topological variants exist – Type I for domains that follow the C1 domain (cPKCs) and Type II for domains that precede C1 (nPKCs). In the calcium-responsive C2 domains of cPKCs, the pocket is lined by multiple aspartic acid residues that coordinate two to three calcium ions that act as a bridge between the C2 domain and the phospholipid head groups of the membrane.CR, cysteine rich; PS, pseudosubstrate; DAG, diacylglycerol; PMA, phorbol-12-myristate 13-acetate; PB1, Phox–Bem 1; C, constant regions; V, variable regions.Box 21977–1987: introducing PKC•Discovery of PKC by Nishizuka and colleagues .•Structural analysis of the PKC isoforms.•2+/phospholipid and DAG  through overlay assay by Jaken .•Compartmentalization of PKC through binding proteins and substrates.•Studies on the temporal kinetics of PKC function.•Elucidation of cell and stimulus-specific actions of PKC isozymes.2008–present: inter-PKC regulation and PKC-signaling moduleThis is the decade where the concept of inter-PKC regulation in calibrating receptor triggered effector functions is gaining popularity leading to the possible build-up of a PKC-signaling network in space-time coordinates.2+-regulated membrane anchor in cPKCs (2+ and instead possess a Phox–Bem (PB)1 domain that facilitates interactions with scaffolding proteins leading to constitutive activation (in vivo may lie in the isoforms’ primary structure and the cofactors governed conformational plasticity. Yet, their promiscuity in vitro suggests involvement of factors beyond the primary structure leading to the studies on the spatio-temporal regulation of these isozymes.Structural reconfigurations of the PKC isoforms play a central role in orchestrating their spatial distribution and activation . C1 domain cPKCs , 15 whilin cPKCs , 19. Thetivation . Phosphotivation . Classictivation . A phospFunctional specificity of the PKC isoforms depends on the proximity to their substrates in specific intracellular compartments effected through multiple binding proteins that also determine the precise duration and amplitude of PKC activity thus providing a mechanism for integrating PKC-mediated signaling with other cellular activities. PKCβI interacts with Bruton’s tyrosine kinase to positively regulate JNK signaling and cytokine gene expression in mast cells , 21. Par2+-induced translocation triggered by the C2 domain is faster than that triggered by the C1 domain (As multiple PKC isoforms may be activated by a single stimulus, it is logical to assume that differential activation dynamics can impose specificity. Following receptor ligation, the isoforms exhibited differences in activation kinetics and subcellular localization. The kinetics of Ca1 domain  (Figure 1 domain  guided b1 domain , 31. Ind1 domain . PKCδ-ac1 domain . These o+ T cells (+ and CD8+ T cells have been observed in mice (de novo IL2 mRNA transcriptional transactivation in primary mouse T cells independently of the actions of PKCθ (Coordination between the concomitantly expressed and activated PKC isoforms within a temporal framework seems to be regulated by transphosphorylation of the isoforms . Followi T cells . In inte T cells . nPKC is T cells . Opposin in mice . PKCα an of PKCθ . PKCϵ ac of PKCθ . These iAcanthocheilonema viteae infection, whereas PKCβ and ϵ act as negative regulators (Mycobacterium tuberculosis-infected macrophages, PKCα upregulates proinflammatory response in conjugation with TLR2 on pretreatment with arabinosylated lipoarabinomannan (via receptor-mediated endocytosis. Infection might cause impairment of Ca2+-host signal transduction, which in turn may affect classical PKC isoforms. Leishmania donovani infection or recombinant IL-10 treatment of macrophages inhibits both the activity and expression of the cPKC isoforms (Leishmania major also impairs PKCα, βI, βII isoforms while enhancing PKCδ and ζ isoforms in macrophages (Leishmania infection (With so many available isoforms, PKC is an ideal candidate for intracellular perturbators Figure C. Reciprgulators . cPKC acgulators . In Myconomannan . While Pnomannan . PKCα denomannan . This isisoforms . Leishmarophages , 37. Incrophages  and concrophages  may causnfection . Comparinfection . Intracenfection , 47, 54.From the evidences gathered, the existence of inter-PKC regulation and a PKC-signaling module seem a logical plausibility. The signaling specificity is generated by the combinatorial assemblies and spatio-temporal dynamics of the PKC isoforms allowing calibration and kinetic modulation of the pathways of the receptor-regulated cellular responsiveness. Although the extreme diversity of PKC responses based on cellular and stimulus differences and the lack of appropriate tools and specific inhibitors pose a major hurdle in building of a generic PKC-signaling map, the analyses provide a conceptual framework placing all PKC isoforms in a single space-time network and a novel principle for devising therapeutic strategies against pathophysiological conditions.AM and SR wrote the manuscript and built the figures. DM and BS critically reviewed the manuscript and made the necessary edits. The final manuscript was a result of the joint efforts of all the authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


WPP domain interacting proteins), which act as protein anchors in the outer NE. The plant KASH homologs are C-terminally anchored membrane proteins with the extreme C-terminus located in the nuclear periplasm; AtWIPs contain a highly conserved X-VPT motif at the C-terminus in contrast to PPPX in opisthokonts. The role of the LINC complex in organisms with a cell wall, and description of further LINC complex components will be considered, together with other potential plant-specific functions.Following the description of SAD1/UNC84 (SUN) domain proteins in higher plants, evidence has rapidly increased that plants contain a functional linker of nucleoskeleton and cytoskeleton (LINC) complex bridging the nuclear envelope (NE). While the SUN domain proteins appear to be highly conserved across kingdoms, other elements of the complex are not and some key components and interactions remain to be identified. This mini review examines components of the LINC complex, including proteins of the SUN domain family and recently identified plant Klarsicht/Anc/Syne-1 homology (KASH) domain proteins. First of these to be described were WIPs ( The nuclear envelope (NE) fulfills many important functions: protecting and enclosing the genetic material, facilitating transport, involvement in cell signaling, and providing physical and structural bridges. These functions of support, transport, and communication are required to different extents in various organisms from single cells to complex metazoans and require a high level of complexity. This is achieved by differentiation of the proteins of the inner and outer nuclear membranes , each having a unique protein composition providing specific cytoplasm-facing and nucleoplasm-facing functions.Communication across the NE occurs through protein bridges that link across the periplasmic space between the INM and ONM. These protein bridges have multiple functions, providing support and anchorage for the genetic material and nucleoskeleton, positioning, and moving the nucleus and acting as a pathway of signaling. While these functions and structures are conserved in eukaryotes, there are marked differences in the proteins that are involved .Micrographs of the nucleus reveal many connections between structural proteins of the cytoplasm and nucleoplasm, the nuclear membrane and the nuclear pores . The memThe LINC complex has two key constituents, the SAD1/UNC84 (SUN) domain proteins of the INM and the Klarsicht/Anc/Syne-1 homology (KASH) domain proteins of the ONM . They atCaenorhabditis elegans embryos and Sad1, a spindle pole body component in Schizosaccharomyces pombe  and Mps3 (Saccharomyces cerevisiae) are located at spindle pole bodies (SPBs) and the INM  with each other than with either ZmSUN1 or 2  suggesting separate gene duplication events. Some difference in function and binding as well as location is suggested for the different SUN proteins. The two plant C-terminal SUN domain proteins are significantly smaller than mammalian and closest in size to yeast Sad1. There is a strong structural resemblance with a single coiled coil domain located between the N-terminal transmembrane domain and the C-terminal SUN domain , Studies of higher plants reveal an additional family of proteins containing a central SUN domain in addition to the C-terminal SUNs . Genome D. melanogaster (Klarsicht), C elegans, . Binding occurs when the PPPT motif of the KASH domain fits into a hydrophobic pocket formed by the SUN domain trimer. Specifically, the penultimate proline appears important for this binding and is widely conserved  in the periplasm ending in a conserved sequence which in most animal KASH proteins is PPPX . KASH doonserved .Syne1/Nesp1 gene that encodes a range of splice isoforms, the biggest being the 1000 kDa Nesprin 1 Giant (Nesp1G). Nesp1G has an N-terminal actin binding domain, made up of two calponin homology domains. Nesprin 2  encodes Nesp2G of 800 kDa also with an N-terminal cytoplasmic actin binding domain  domain proteins. These include WPP1, WPP2, and RanGTPase-activating protein 1 (RanGAP1) and are characterized by the presence of various repeats of the WPP motif in the protein sequence (WPP domain interacting proteins) and the WITs (WPP interacting tail anchored proteins). WIPs and WITs oligomerise to provide the anchorage of the WPP proteins. For instance, by anchoring RanGAP1 to the NE (Previous work  had idensequence . These ho the NE , they aro the NE . This mosun1-KO/ sun2-KD mutant transformed to express GFP-AtWIP1, the fluorescent signal from the WIP protein was predominantly cytoplasmic, resembling the distribution shown for a WIP1 truncation mutant in which the C-terminal VVPT had been deleted, confirming the requirement for the SUN domain protein for nuclear localisation. Comparison of the AtWIP1 C-terminus shows a low degree of similarity to opisthokont KASH domains. It is small in size, though similar to C. elegans ZYG-12B and KDP-1 and D. discoideum Interaptin (Confirmation of the WIPs as KASH domain proteins depended on demonstration of their SUN interaction and that the SUN binding was indispensable to their NE localisation. Interaction of the VVPT motif with SUN domain proteins was shown by a number of means. Deletion of all, or parts of, the SUN domain abolished interaction of AtWIP1, 2, and 3 in a pull down assay with AtSUN2 . Also, fteraptin . The penbona fide function identified for a plant LINC complex; it differs from mammalian systems, where RanGAP anchorage occurs by direct attachment to the nuclear pores by RanBP2. Further functions of the plant LINC complex are being elucidated. The location of RanGAP at the pore complex was the first In fact, a very recent study by Other interactions at the plant NE seem to be implicated in the interaction between the nucleus and microtubule cytoskeleton. The mini review of A role for the LINC complex in both nuclear shape and movement indicates this is a highly conserved and fundamental system of nuclear attachment. In the absence of a variety of other NE proteins found in ophistokonts, the plant SUN and KASH proteins take on an increased importance. Future work to elucidate the functions of the C-SUNs and the mid-SUNs, together with identification of further plant KASH proteins and exploration of their roles is of great importance as the system is central to many key plant cell processes from control of gene expression to chromosome segregation in meiosis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate , which is involved in B-cell development. This enzyme is usually kept inactive until the B-cells are needed, but the molecular mechanism of this restraint remains to be discovered.Wang et al. have used X-ray crystallography and biochemistry to produce a three-dimensional atomic model of the Btk enzyme in its inactive form. The model and functional studies reveal that when the Btk enzyme is inactive, it folds up into a compact shape. Within the enzyme, a ‘PH-TH module’ and two other domains work together to stabilize this folded-up form.6 (short for inositol hexakisphosphate) can activate the Btk enzyme even in the absence of a membrane. This molecule recognizes a previously unnoticed binding site on the PH-TH module, and causes the PH-TH module in the inactive Btk enzyme to bind briefly to the PH-TH modules in other Btk enzymes. This brief binding event is an important step in the process that activates the enzyme. IP6 might also be involved in recruiting the Btk enzyme to the cell membrane in B cells, but this proposal needs to be tested experimentally. The details of the BTK activation mechanism reported by Wang et al. could prove useful to researchers developing new treatments for immunodeficiency diseases.Although earlier studies indicated that the Btk enzyme becomes activated only when it is recruited to cell membranes, Wang et al. found that a small molecule called IPDOI:http://dx.doi.org/10.7554/eLife.06074.002 Activation of B- and T-lymphocytes relies on a chain of tyrosine phosphorylation events initiated by B-cell and T-cell receptors, respectively . These p3). Btk dysfunction is the cause of X-linked agammaglobulinemia, a severe disease of primary immunodeficiency , in solution. A flexible linker connects the PH-TH module to the Src-like module; its length varies from 26 residues in Itk, the Tec family kinase in T-cells, to 45 residues in Btk.The Tec family kinases have a lipid-interaction module attached to the N terminus of a Src-like module  . Like thTyrosine phosphorylation controls Btk activity. B-cell activation leads to rapid phosphorylation of Tyr 551 in the kinase domain and Tyr 223 in the SH3 domain of Btk . Tyr 5513, produced as a consequence of receptor activation, provides a simple control mechanism for switching on the kinase. Membrane recruitment triggers trans-autophosphorylation, through increased local concentration, or trans-phosphorylation by membrane-bound Src-family kinases, thereby activating the Tec kinase , a soluble signaling molecule ubiquitous in animal and plant cells  and the isolated kinase domain with mutations in the activation loop (determined at 1.6 Å). The details of the crystallographic experiments are given in We have determined four crystal structures as part of this work. Two of these structures help explain how Btk is autoinhibited. These are the structures of the Btk SH3-SH2-kinase module (determined at 2.6 Å resolution) and of the PH-TH-kinase unit (determined at 1.7 Å resolution). The other two structures are of the isolated PH-TH module bound to IPWe determined the structure of a construct of mouse Btk spanning residues 214 to 659, which starts just before the SH3 domain and ends at the C terminus of full-length Btk. The structure resembles those described previously for autoinhibited, Src-like, non-receptor tyrosine kinases, with the SH2-kinase linker sandwiched between the SH3 domain and the small domain of the kinase (the N lobe of the kinase) . The cryThe sequence identity between Btk and c-Abl is 42%, 31%, and 46% in the SH3, SH2, and kinase domains, respectively; for Btk and c-Src, it is 44%, 29%, and 40%. Superposition of the C lobes of the Btk and c-Abl kinase domains gives an approximately 20° relative rotation of their SH3 domains; superposition of the SH3 domains, a relative rotation of about 15° for their SH2 domains. Similar results are obtained for a comparison to c-Src. Coupling between the SH3 and SH2 domains may therefore be somewhat different in these kinases .The structure of the SH3 domain of Btk is nearly identical to those determined previously using NMR (the r.m.s. deviation for 53 Cα atoms is 0.9 Å) , except Diffuse electron density for the SH2 domain at every stage of our structure determination shows that this part of the polypeptide chain occupies a range of positions and adopts multiple local conformations. The domain itself has a marginally stable internal structure, as shown by the domain swap, and it is probably imperfectly locked in place. The SH2 βA/αA loop lies between kinase-domain helices αB and αF. Density features for this loop are relatively sharp, and the rest of the domain may pivot somewhat about its contact with the C lobe.The domain swap occurs in the same location within the SH2 domain as a domain swap seen in a Grb2 SH2-domain crystal structure . The SH2There is no evidence that a domain-swapped dimer  is a functional state of intact Btk, and so it is likely that the assembled state involves a single polypeptide chain, rather than two inter-folded chains. The relatively high concentration of purified protein could have promoted dimer formation in our preparations. In the case of Grb2, the structure of the intact protein, a monomer in solution, shows no domain swap, and loss of contacts between the deleted N- and C-terminal SH3 domains can account for destabilization of the central SH2 when it is prepared as an isolated domain . For BtkThe unphosphorylated activation loop of Btk folds into the mouth of the catalytic cleft, blocking part of the substrate-binding site, and helix αC lies at the margin of the N lobe, preventing formation of a Glu-Lys salt bridge conserved in all catalytically active protein kinases  Figure . The actA network of polar interactions present in the autoinhibited forms of c-Src and Hck is also present in the Btk conformation in our crystals . Glu 445The structure just described demonstrates that the SH3-SH2 domain in a Tec-family member can assemble into a regulatory clamp, just as it does in other Src-like kinases. In the crystal structures of c-Src, Hck, and c-Abl, a latch is present—either in the form of a phosphorylated C-terminal tail  or a myristoyl group bound in a pocket on the C lobe of the kinase domain (c-Abl). In both cases, the latch secures the SH2-domain/C lobe contact, precisely the interface that appears to be poorly formed in our Btk crystals. As we show below, the latch in Btk is provided by the PH-TH module.We expressed a ‘PH-TH-kinase’ construct of bovine Btk that contains the PH-TH module connected to the kinase domain by the 13-residue SH2-kinase linker. We replaced six residues in the activation loop of Btk with the corresponding ones in Itk, shown previously to stabilize the activation loop of the Btk kinase domain . This imThe overall conformation of the kinase domain, as well as the specific conformation of the activation loop, is very similar in the two structures we have determined . We alsoThe PH-TH module contacts the kinase N lobe. The interface differs substantially from the one found in Akt1, another PH-domain regulated kinase  Figure . In Akt1We combined the two structures just described to build a model for full-length Btk . AligninIn simulations of the SH3-SH2-kinase module, the initial and final conformations of the SH3 and kinase domains had r.m.s. deviations in Cα positions of only ∼3.0 and ∼2.0 Å, respectively, after first aligning the C lobe of the kinase domain . The SH2In simulations of the PH-TH-kinase construct, the interface with the kinase domain remained intact, but the PH-TH module wobbled back and forth through a rotation of about 20° . The aveeLife website , as determined by mass spectrometry, is consistent with the calculated molecular weight .2+ initiates autophosphorylation, leading in turn to increased catalytic activity. We monitored activation in two ways. First, we monitored the phosphorylation by Btk of a peptide substrate derived from PLC-γ2, using a continuous kinase-coupled colorimetric assay . SUVs containing 75% DOPC, 20% DOPS, and 5% PIP3 produce a rapid and substantial activation of Btk .We studied the effect on Btk activity of PIPn of Btk . At a Btrylation . An esti3 is IP4, and so this was included in our study, as were several other inositol phosphates  promotes Btk autophosphorylation strongly at 2 μM protein concentration, substantially enhancing Btk catalytic activity within the first 5 min after addition (5), but these required higher protein concentration and longer times for appreciable autophosphorylation than did IP6 , full-length Btk is activated within the first 5 min of reaction time, in contrast to the 30 min and 120 min that are required for the activation of the SH2-kinase module and full-length Btk, respectively, in the absence of IP6 or PIP3-containing vesicles  activates only slightly faster than full-length Btk in the absence of IP6. Nevertheless, in order to test this possibility, we introduced two mutations (Y133E and R134E) at the PH-TH-kinase interface that are expected to disrupt the inhibitory docking of the PH-TH module on the kinase domain. Full-length Btk bearing these two mutations retains activation by IP6, showing that IP6 does not act by dislodging the interactions seen in the crystal structure  is located ∼15 Å from the kinase-interacting surface of the PH-TH module  to study the interaction between IPs of Btk . The ITCf 250 nM . We intr binding . IP4 binle level . The ITC6 to the IP4-binding deficient mutant is relatively weak, the observed affinity is consistent with the EC50 value of 20 μM for IP6 activation, as determined in our activation assays P5 and IP5, have a mildly activating effect on Btk; they are more effective than IP5. These comparisons indicate an important role in Btk activation for the phosphate at position 2  requires the formation of a multi-subunit co-repressor complex, in which InsPtogether . The mec6 arises from its ability to promote dimerization of the PH-TH module, fusion of a Btk PH-TH module with the catalytic domain of another kinase that autophosphorylates slowly should impart activation by IP6. A good example of such a kinase is c-Abl. Indeed, in chronic myelogenous leukemia, a chromosomal translocation results in fusion of a coiled-coil oligomerization domain with c-Abl, leading to hyper-activation of the BCR-Abl fusion protein by autophosphorylation. Several other fusion variants involving c-Abl, in which different oligomerization domains are present, are associated with hematological malignancies (If the effect of IPgnancies .6 at a saturating concentration (100 μM). The fusion protein at a concentration of 1 μM autophosphorylated slowly in the absence of IP6, while the IP6-bound protein became heavily phosphorylated within the first 30 s, the dead-time of our assay  presumably drives the formation of the Saraste dimer, which is otherwise not favored.Our failure to detect stable dimers of the Btk PH-TH module or of full-length Btk in the presence of IP6 nor IP4. Within the first 30 ns of simulation, the β3-β4 hairpin bent towards the core of the PH-TH module , to reduce the positive charge on the protein by four units. The autophosphorylation rate of full-length Btk with the K49E/R52E mutations was substantially higher than that of the wild-type protein, with pronounced phosphorylation observed in the first 2 min of the reaction  was used to prepare baculovirus. Infected Sf21 cells were harvested by centrifugation 72 hr post-infection, then frozen in liquid nitrogen for storage. Cells were later thawed, resuspended in 25 mM Tris·Cl, pH 8.0, 50 mM NaCl, 5 mM DTT, 5 mM benzamidine, and complete protease inhibitor tablets , and lysed mechanically by Dounce homogenization followed by brief sonication. All remaining steps were carried out at 4°C. Cellular debris was cleared by centrifugation at 96,000×Fractions containing the Src-like module of Btk were identified by Western blot, pooled, brought to 0.7 M ammonium sulfate, then loaded on to a phenyl sepharose column pre-equilibrated with 50 mM Tris·Cl, pH 8.0, 0.7 M ammonium sulfate, and 5 mM DTT. Contaminating protein was removed by an initial step to 0.2 M ammonium sulfate and the column eluted with a linear gradient to 50 mM Tris·Cl, pH 8.0 and 5 mM DTT over four column volumes. The Src-like module of Btk eluted as a broad peak starting at approximately 0.18 M ammonium sulfate.Fractions containing significant amounts of the Src-like module of Btk, identified by SDS PAGE, were pooled and concentrated using Centricon Plus-80 concentrators . Concentrated protein was dialyzed against 4 × 1 liter changes of 25 mM Tris·Cl, pH 8.5, 25 mM NaCl, 5 mM DTT. That buffer, excepting changes to the NaCl concentration, was maintained through the rest of the purification. After dialysis, protein was loaded on to a mono-Q HR 10/10 column, and eluted with a linear gradient from 50 mM NaCl to 250 mM NaCl over twenty column volumes, with the protein eluting as two peaks at approximately 100 mM NaCl (peak A) and 150 mM NaCl (peak B), respectively. Fractions from the two peaks were pooled separately. Fractions from peak B were dialyzed against buffer containing 20 mM NaCl. The two pools could then be concentrated using Centricon-10 concentrators (Millipore). They were loaded separately onto a HiPrep S-200 16/60 size exclusion column pre-equilibrated in buffer containing 25 mM NaCl. Peak A eluted at the position corresponding to a globular protein of molecular mass 55 kDa, the value expected for a monomer of the Src-like module. Peak B eluted at a position corresponding of 140 kDa, compatible with an extended dimer. The protein in peak A could be concentrated using Centricon concentrators to approximately 20 mg/ml, while peak B required dialysis against a buffer containing no more than 10 mM NaCl, followed by concentration in dialysis tubing laid on PEG 20,000 at 4°C. In this way, the dimeric protein could be concentrated to approximately 10 mg/ml.Peaks A and B were definitively identified as the Src-like module of Btk monomer and dimer, respectively, by chemical cross-linking using disuccinimidyl glutarate  and PAGE analysis (results not shown). All column media and pre-packed columns were purchased from Amersham Biosciences.2, 50 mg/l Kanamycin, 50 mg/l streptomycin, and 50 mg/l chloramphenicol. After 16 hr, cells were harvested by centrifugation, resuspended in Ni-NTA buffer A , and flash-frozen in liquid nitrogen. Cells were then thawed on ice and supplemented with a mixture of protease inhibitors .DNA encoding bovine Btk (residues 1 to 659) was cloned into pET-28 expression plasmid yielding N-terminally 6-histidine-tagged SUMO-fusion protein. The hexahistidine-SUMO tag was cleavable by Ulp-1 SUMO-specific protease. For protein expression, the plasmid was transformed into BL21 DE3*  cells that also contain the expression plasmids for GroES/GroEL and phosphatase YopH , and theAfter cell lysis by French press and removal of cell debris by centrifugation, clear lysates were loaded onto a HisTrap Ni-NTA column , washed with 100 ml Ni-NTA buffer A. Proteins were eluted with a single step of Ni-NTA buffer A supplemented with 250 mM imidazole. Proteins were buffer exchanged into storage buffer  by FastFlow desalting column , and affinity tags were removed by incubation with Ulp-1 protease at 4°C overnight. Cleaved proteins were collected in the flow-through during Ni-NTA affinity chromatography and were subjected to size-exclusion chromatography on a Superdex 200 column  equilibrated in storage buffer. Proteins were concentrated on an AmiconUltra centrifugal filter device  to a final concentration of ∼400 μM. Protein aliquots were frozen in liquid nitrogen and stored at −80°C.The Src-like module (residues 217 to 659), SH2-kinase module (residues 270 to 659), and the kinase domain (residues 394 to 659) of bovine Btk, various bovine Btk PH-TH-kinase constructs and the PH-TH-Abl fusion construct (BTK residues 1 to 216 fused to human c-Abl residues 140 to 518) were all expressed and purified using the same protocol. The PH-TH module of bovine Btk (residues 1 to 172) was prepared in the same way as described above expect that the co-expression of GroEL/GroES and YopH is not necessary.The crystals of the dimeric protein (peak B) were grown using hanging-drop vapor diffusion at 4°C by mixing purified protein at 5 mg/ml in 25 mM Tris·Cl, pH 8.5, 10 mM NaCl, 5 mM DTT with an equal volume of the precipitant solution, 100 mM Tris·Cl, pH 8.5, 200 mM Na·acetate, 7.5% PEG 4000. Wedge-shaped crystals grew to approximately 0.1 mm on a side after a period of 2 weeks. These crystals could be frozen by first transferring them, in four sequential steps, to a solution of 100 mM Tris·Cl, pH 8.5, 200 mM Na·acetate, 15% PEG 4000, followed by transfer in six sequential steps to a solution containing 100 mM Tris·Cl, pH 8.5, 200 mM Na·acetate, 15% PEG 4000, 30% glucose. The monomeric protein (peak A) did not yield crystals.2, 1 mM dimercuriacetate (DMA), 3 mM Au(NO3)4, and no adduct, respectively , beamline A1. Anomalous data, when present, were collected by the inverse beam method. Four sets of data contributed to the structure determination, collected from crystals in 5 mM KAu(CN)ectively . A heavyng SHARP , and difng SHARP .The kinase domain was built into the density modified maps with reference to the already determined Btk kinase domain structure (PDB ID 1K2P), after separately placing the large and small lobes. The SH3 domain was built by comparison with the c-Src SH3 domain (PDB ID 2SRC).2, DMA, Au(NO3)4 and native, using the program DMMULTI . This model was placed by hand, aligning helices αA and αB into the unambiguous density, then refined as a rigid-body using a 4.0 Å resolution cutoff. Multiple crystal averaging across four distinct data sets (KAu(CN) DMMULTI , revealeThe structure was refined using alternating cycles of manual rebuilding in the program O  and tors3 (Avanti lipid) and inhibitor CGI1746 , and was incubated at 4°C for 30 min before setting up trays. IP3 binds to the protein, but does not activate it. Although present in the crystallization mix, it is not visible in the final electron density (see below). Needle clusters of crystals were obtained by sitting drop vapor diffusion by mixing equal volume of protein solutions at 15 mg/ml with reservoir solution containing 200 mM NaCl, 50 mM MgCl2, and 20% PEG3350. To prepare seeds for microseeding, needle clusters were harvested and combined from 20 drops and transferred into a 1.5-ml eppendorf tube.The bovine PH-TH-kinase construct (residues 1 to 170 connected to residues 384 to 659) was thawed on ice and mixed with twofold excess IP2, and 20% PEG3350 were mixed and were equilibrated in a hanging drop tray for 24 hr at 4°C. Microseeding was then performed by striping the wild-type crystal seeds using a cat whisker into the equilibrated drops. Large rock-like crystal clusters appeared ∼4 days after micro-seeding, and single crystals  can be isolated. For cryo-protection, crystals were soaked in reservoir solution supplemented with 25% glycerol, and flash-frozen in liquid nitrogen and stored at 100 K.The bovine PH-TH-kinase construct with activation loop mutations  was prep3 is seen in difference electron density maps, even at low contour levels (2σ above the mean in σ weighted |Fo| − |Fc| difference maps). Data collection and refinement statistics are summarized in Data were collected at the Lawrence Berkeley National Laboratory, Advanced Light Source (ALS), beamline 8.2.1. Data reduction was carried out with the software package HKL2000 . The str6 (Sigma) for 10 min. Plate-like crystals were obtained by sitting drop vapor diffusion by mixing equal volume of protein sample solution and the reservoir solution containing 25% PEG1500 and 0.1 M DL-malic acid pH 5.5. Crystals were then transferred to cryo-protection solution, which is the reservoir solution supplemented with 30% glycerol and 3 mM IP6.The bovine Btk PH-TH module (residues 1 to 172) sample was thawed at room temperature and was incubated with fivefold excess IP6 was generated using REEL in PHENIX (6 seen in the structure of Auxin receptor TIR1 (PDB: 2P1P) , beamline 8.2.1. Data reduction was carried out with the software package HKL2000 . The strn PHENIX  based onB: 2P1P) . Data co6 (Sigma) for 10 min. Crystals were obtained by sitting drop evaporation methods by mixing equal volume of protein sample solution and the reservoir solution containing 0.2 M ammonium citrate dibasic and 20% PEG 3350. Crystals were then transferred to cryo-protection solution which is the reservoir solution supplemented with 30% glycerol and 3 mM IP6.Btk kinase domain (residues 395 to 659) with the activation loop mutations   was thaw6 in crystals. Data collection and refinement statistics are summarized in Data were collected at the Lawrence Berkeley National Laboratory, Advanced Light Source (ALS), beamline 8.2.1. Data reduction was carried out with the software package HKL2000 . The strBtk samples were diluted to 4 μM using the buffer that contains 150 mM NaCl, 25 mM Tris, pH 7.5, and 5% glycerol. For experiments involving lipid vesicles and inositol phosphates, additional 500 μM lipid and 200 μM inositol phosphates are supplemented into the dilution buffer, respectively, followed by a 10-min incubation at room temperature.2, 2 mM ATP, 25 mM Tris, pH 7.5, 2 mM sodium vandate, and 5% glycerol and incubated at room temperature. The final concentration of Btk samples is 2 μM. At each time point, the samples were either frozen in liquid nitrogen and stored at −80°C for the coupled kinase assays, or mixed with equal volume of quench buffer that contains 2X SDS-PAGE buffer supplemented with 100 mM EGTA, followed by incubating at 95°C in a heat block for 15 min for western blot assays.The diluted samples were mixed with equal volume of the reaction buffer that contains 150 mM NaCl, 20 mM MgCl2, 25 mM Tris, pH 7.5, 2 mM sodium vandate. The protein concentrations were kept at 1 μM for all Btk constructs. The peptide sequence is ERDINSLYDVSRMYVDPSEIN, which was derived from residues 746 to 766 of PLC-γ2. The Km value for phosphorylated full-length Btk for this peptide is 925 ± 117 μM, and the peptide concentration was kept at 1 mM in all experiments. The reactions were performed in a 96-well plate and were monitored by SpectraMax . The ATP turn-over rate (v0: min−1) was calculated by fitting the data acquired from first 150 s using a linear regression model.A continuous pyruvate-kinase coupled assay was performed to measure the kinase activity of the proteins as described , with miThe levels of autophosphorylation of purified Btk samples were monitored using non-specific anti-phosphortyrosine antibody 4G10 (EMDMillipore). The total amount of Btk was monitored using coomassie-blue stained SDS-PAGE. For each set of experiments, 10 μl of each Btk sample was loaded on two 12% SDS-PAGE gels. The two gels were run at 250 V, 400 mA for 35 min at room temperature.One gel was transferred to 50 ml fixing buffer containing 50% ethanol and 10% acetic acid and was heated for 30 s in a microwave. The gel is then transferred to 50 ml staining buffer that contains 5% ethanol, 7.5% acetic acid, and 0.00025% coomassie brilliant blue R-250 and was heated for another 30 s in a microwave. The gel was then left to cool at room temperature in the staining buffer on a rocker for 1 hr and was then imaged using ImageLab .The other gel was transferred into western blot transfer buffer that contains 25 mM Tris at pH 7.4, 192 mM glycine, and 20% methanol and was incubated at room temperature for 15 min. Protein samples were then transferred to PVDF membranes from the gel using the semi-dry transfer device TRANS-BLOT (Bio-Rad). The membranes were then blocked for 1 hr in TBST buffer  supplemented with 5% dry milk, and incubated with primary antibody 4G10 (1:2000 dilution) at 4°C overnight. The membranes were then washed three times in TBST buffer, and incubated with HRP-linked anti-mouse IgG antibody (1:5000 dilution) in TBST buffer at room temperature for 1 hr. The membranes were then washed three times in TBST buffer, incubated with 0.5 ml WesternBright Quantum  for 1 min, and were imaged using ImageLab (Bio-Rad). The western blots were reproduced at least three times and the most representative ones were chose to present.3  were dissolved in chloroform and stored at −20°C. For liposome preparations, the three lipids are mixed in a test tube at the desired molar ratio (10:75:5); chloroform was evaporated under a nitrogen atmosphere for 15 min; the samples were transferred to a vacuum desiccator and dried overnight at room temperature. Dry films were hydrated in buffer containing 25 mM Tris-HCl (pH 7.4) and 100 mM NaCl to a concentration of 10 mg/ml.Three lipids, 18:1 DOPS, 18:1 (Δ9-Cis)(DOPC), and 18:1 PI PThe hydrated liposomes are heterogeneous in size and the diameters of the liposomes vary from ∼50 nm to ≥ 1 μm. To prepare small unilamellar vesicles, hydrated liposomes were subject to seven freeze–thaw cycles using liquid nitrogen, followed by extrusion through 100-nm filters . The liposomes prepared using this method have diameters around 100 nm, as measured using negative staining electron microscopy (data not shown).4 and IP6 were diluted using the same buffer to a final concentration of 300 μM. Each set of ITC experiments included three samples: 420 μl of Btk protein sample in the cell, 150 μl of IP4 or IP6 at a concentration 300 μM in the syringe, and 500 μl of binding buffer. All samples are stored at 4°C before the titration experiments.The isothermal titration calorimetry experiments were performed using MicroCal-autoITC 200 . Various Btk samples are diluted in the binding buffer containing 150 mM NaCl, 25 mM Tris, pH 7.5, and 5% glycerol to a final concentration of 20 μM. IPa and binding enthalpy, ΔH. The binding entropy is then calculated using formula: ΔS = (ΔH + RTlnKa)/T. Thermodynamic parameters for various Btk constructs binding to IP4 and IP6 are listed in The ITC experiments were performed at 20°C. An initial injection of 0.5 μl was excluded from data analysis, followed by 14 injections of 3 μl each, separated by 180 s with a filter period of 5 s. The protein solution was stirred at 500 rpm over the course of titration. Titration curves were fit with a one-site binding model. The three fitting parameters are stoichiometry, N, association constant, K+ and Cl−) were added to neutralize the net charges. After initial energy minimization, the systems were subjected to 100 ps of constant number, volume and temperature (NVT) equilibration, during which the system was heated to 300 K. This was followed by a short equilibration at constant number, pressure, and temperature . Finally, the production simulations were performed under NPT conditions, with v-rescale thermostats in Gromacs 4.6.2, respectively, in the absence of positional restraints. Periodic boundary conditions were imposed, and particle-mesh Ewald summations were used for long-range electrostatics and the van der Waals cut-off is set at 10 Å. A time step of 2 fs was employed and the structures were stored every 2 ps. His 143, Cys 154, Cys 155, and Cys 165, which coordinate Zn2+ in the PH domain were set to be deprotonated. No additional positional restrains are applied to the Zn2+ ion, and its sp3 tetrahedral coordination remains intact during all MD simulations.Molecular dynamics trajectories were generated using the Gromacs 4.6.2 package  using thThe visual molecular dynamics (VMD) analysis toolkit  was usedThe active conformation of the Btk kinase domain was modeled based on that of the Lck kinase domain (PDB: 3LCK) , using MC) of Btk on a lipid vesicle by the following equation:We estimated the effective concentration  are bound to Btk, then,N0 is the total number of lipids on a vesicle. This is given by,A0 is the surface area of a lipid molecule. We assume all lipids have the same surface area for their headgroups, which is roughly 0.6 nm2 . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate” for consideration at The Reviewing editor and two other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.6. Btk is of broad interest in immune signaling and as a drug target. This paper enlightens the community about several very interesting and important features regarding Btk regulation: 1) The kinase domain is held in an inhibited state by the SH2 and SH3 domains in a manner analogous to Src (and Abl);The manuscript by Qi Wang et al concerns a comprehensive biochemical analysis of the regulation of Btk (Bruton's tyrosine kinase) and its unexpected activation by IP2) Unique to Btk, its PH domain participates directly through the kinase N-lobe with the SH2 and SH3 domain to suppress the kinase domain;3 can activate Btk by inducing dimerization;3) Vesicle bound PIP6 can activate Btk by binding a non-canonical surface of the PH domain and promoting dimerization.4) Soluble IP6 regulation is perhaps the most special aspect of the manuscript and should generate substantial interest in the tyrosine kinase and pleckstrin homology domain fields. While the authors rightly cite Huang et al, Science (2007),which reports that soluble IP4 can activate ITK tyrosine kinase, our read of this study is that it minimally characterizes this putative activation, which in vitro was quite marginal. In summary, the current manuscript is an excellent fit for eLife. We ask the authors to consider the comments below.The comprehensive model of Btk regulation described in this manuscript derives from several high-resolution crystal structures, enzymologic analysis of autophosphorylation as well as peptide phosphorylation in the presence of various activators, site-directed mutagenesis, and molecular dynamics simulations. The discovery of IPThere is no mention in the Introduction of the prior Btk structures reported. A search of the PDB reveals that there are over 30 structures of various Btk domains in active and inactive states. It would be helpful to the reader to briefly summarize the known Btk domain structures and include a statement indicating that although the structures of the individual Btk domains are established, the relationships between the kinase domain and the SH2, SH3 and PH-TH domains were not known prior to the current manuscript.Major comments:6 as a glue holding neighboring PH domains together is somewhat reminiscent of a related role for soluble IP4/6 in stabilizing HDAC/corepressor complexes . The authors may wish to compare the Btk and corepressor inositol phosphate-containing structures and comment on the relationship if appropriate.The role of IP6 rather than PIP3 activation, this could give rise to different cellular signaling outputs.We were unable to find the specific peptide sequence of the kinase substrate or its concentration in the Methods. While not absolutely essential, it would also be informative to know what the Km of the peptide is and whether this changes in different states of Btk activation. If there were Km differences among particular Btk conformational states, for example IP6 or PIP3 binding? Likewise, is there any reason to think that ligation of the SH3 domain by external ligands impacts this SH3/PH interaction?In their full-length Btk model, the SH3 domain and the PH domain appear to make electrostaic interactions with each other . Do the 3-derived IP4 binds more tightly to the Btk PH domain than IP6 (Kd of 30 nM vs. 250 nM), it is interesting to consider that high concentrations (>20 µM) of soluble IP6 could conceivably compete Btk off of the PIP3 containing membrane. Thus, one way to consider the importance of the Btk allosteric binding site for IP6 is that it is needed to prevent down-regulation by competition of IP6 for PIP3 at the PIP3 binding site. If true, Btk membrane binding may be an overstated facet of its regulation, as the authors also suggest. It will be fascinating in future studies to investigate selective mutants in vivo to dissect the relative contributions of PIP3 and IP6 in signaling.Although the authors show that PIP6 might stimulate dimerization of membrane-tethered Btk, as suggested in the Conclusions, might it not also enhance the affinity (avidity) of Btk for PIP3-containing membranes by promoting dimerization? This would have the effect of 'sensitizing' Btk to PIP3 levels, and could be important. Such an effect of IP6 (or IP7 or IP8) could also/alternatively counteract inhibition of PH domain function by competition at the canonical site . It might thus selectively 'spare' Btk from such inhibition?Alternatively, just as IPIn the beginning of the Results and Discussion section, the authors describe the difference in the angles for the kinase SH3 domain with respect to other kinases and they explain the differences in orientation of the SH2 and SH3 domains, saying: “Coupling between the SH3 and SH2 domains may therefore be somewhat different in these kinases.” Although this may be true, it is also possible that there is not a fixed orientation in solution between the different domains and the differences reflect different arrangements captured by the crystals. The authors also say that: ”diffuse electron density for the SH2 shows that this part of the polypeptide chain occupies a range of positions and adopts multiple conformations“. This statement seems to support such an idea.Also in the Results and Discussion section, the authors say that: “… nearly identical to those determined previously using NMR”. They should include RMSD for the C-alphas that were aligned.This sentence, in the same section of the text: “The domain swap occurs in the same location within the SH2 domain as a similar swap seen in a Grb2 SH2-domain crystal structure.” From the drawing this similarity is not so apparent. They use the same helix against the same beta sheet, but the orientation of the two monomers appears different. In Grb2 the helices are essentially parallel to the beta-sheet whereas in Btk, these helices seem to be perpendicular.The authors say that it is unlikely “that a domain-swapped dimer” is physically important. That being the case, the authors may wish to consider reducing the overall discussion about domain swapping.In the Results and Discussion, it says that “r.m.s. deviation from c-Src is 2.3 Å for the 24 α-carbons”. This seems to be a rather large deviation for so few carbons.In that same section of the article, the authors call an Arg-Phe interaction an “amino-aromatic interaction”. Is this actually amino (cation)-aromatic pair, or is it a π-type (Pi-type) interaction between the guadinium of the Arg and the aromatic ring of the Phe?In the subsection “Structure of a PH-TH-kinase construct of Btk”, it says: “The PH-TH module contacts the kinase N-lobe. The interface differs from the one found in Akt1”. Is this because they are intrinsically different or because of the deletion of the SH2 and SH3 domains? Some comment on this could be helpful.Subsection headed “A structural model for full-length Btk”: it is not very clear what the cause is, but the deviations between the crystallographic structures and the ones at the end of the trajectories appear to be large. It is not very clear what these trajectories were probing and what the implications are for their having departed so significantly from the original structures?6 in solution” section: “but these required higher protein concentration and longer times for appreciable autophosphylation than did IP6IP6.” Looking at 5 doesn't show higher protein concentration.The manuscript says, in the “Activation of Btk by IP6-mediated Btk activation”: when the authors note that R28C/N24E abolishes IP4 binding and weakens IP6 binding by 20-40-fold using ITC, they appear to conclude that the residual binding is solely due to the separate IP6 site. Is it possible that the double mutant still allows for IP6 binding to the IP4 site? Perhaps the authors can clarify this section a bit more.Subsection headed “The canonical lipid-binding pocket of the PH-TH module is not critical for IP6-bound PH-TH module”: it is not said, although one can assume, that the dimers are two-fold symmetric. If that is the case it is not very clear whether or not the open-ended chain is a consequence of the crystal lattice rather than being biologically relevant. Therefore, the binding site that is formed by two molecules in the context of the open-ended chain of dimers may not be present in solution. The authors appear to imply that the IP6 interaction site is mostly determined by one of the chains. If that were the case and IP6 binds to that site even in the absence of the dimer-formed open-ended chain, then shouldn't one have observed a more complex stoichiometry in the ITC experiments (which shows an n∼1)? More explanation here could be helpful.Subsection headed “Structure of the IP6 regulation is fairly lengthy given the degree of speculation. The authors could consider condensing this.The section on IPIn the beginning of the Results and Discussion, it could be helpful to include a short introduction indicating which structures were determined as part of this work, to what resolution, and a reference to 6 in the peripheral site is well defined or not. IP6 binding to such a 'flat' (as the authors put it) site is often heterogeneous in pose, but the data in 6-mediated Btk activation”. Some comment on stereospecificity would be helpful for those who think about IP6 binding sites. Perhaps a density panel could be added to It would be interesting to know whether the orientation of the IPIn reflecting on the organization of the paper, one suggestion would be to place  There is no mention in the Introduction of the prior Btk structures reported. A search of the PDB reveals that there are over 30 structures of various Btk domains in active and inactive states. It would be helpful to the reader to briefly summarize the known Btk domain structures and include a statement indicating that although the structures of the individual Btk domains are established, the relationships between the kinase domain and the SH2, SH3 and PH-TH domains were not known prior to the current manuscript.We appreciated this point. We have added two sentences in the Introduction summarizing the previously determined structures of Btk fragments and have prepared a supplemental table  that lisMajor comments:The role of IP6as a glue holding neighboring PH domains together is somewhat reminiscent of a related role for soluble IP4/6in stabilizing HDAC/corepressor complexes . The authors may wish to compare the Btk and corepressor inositol phosphate-containing structures and comment on the relationship if appropriate.4 in regulating the HDAC/corepressor complex, and compared that with the role of IP6 in regulating Btk activation. The mechanism of IP6-induced transient Btk dimerization is fundamentally different from that of the regulation of HDACs by IP4. We point out in the revised manuscript that in case of Btk, the peripheral IP6 binding site is located next to the Saraste dimer interface, and IP6 is not directly involved in any interactions between the two Btk subunits in the Saraste dimer.We have added a paragraph in the Results section discussing the role of IPWe were unable to find the specific peptide sequence of the kinase substrate or its concentration in the Methods. While not absolutely essential, it would also be informative to know what the Km of the peptide is and whether this changes in different states of Btk activation. If there were Km differences among particular Btk conformational states, for example IP6rather than PIP3activation, this could give rise to different cellular signaling outputs.We apologize for not including this information in the original manuscript. We have added the peptide sequence and the Km value for the peptide for phosphorylated full-length Btk in the Materials and methods section. We did not measure the Km values for various Btk states, and we agree that this could be an interesting subject for the future studies.In their full-length Btk model, the SH3 domain and the PH domain appear to make electrostaic interactions with each other . Do the authors predict that this surface is modulated by IP6or PIP3binding? Likewise, is there any reason to think that ligation of the SH3 domain by external ligands impacts this SH3/PH interaction?6 site with respect to other regulatory elements. We don’t think that IP6 or PIP3 binding will directly disassemble the inactive conformation of Btk, because both binding sites are located distal to the interface between the PH-TH module and the Src-like module. We have clarified this by marking both binding sites in our cartoon illustrations in all figures and have added corresponding sentences in the Results section. On the other hand, the ligation of the SH3 domain by external ligands will likely disassemble inactive conformation of Btk. We now note this in the Results section.This point made us realize that we had not provided structural diagrams that clearly showed the location of the peripheral IPAlthough the authors show that PIP3-derived IP4binds more tightly to the Btk PH domain than IP6(Kd of 30 nM vs. 250 nM), it is interesting to consider that high concentrations (>20 µM) of soluble IP6could conceivably compete Btk off of the PIP3containing membrane. Thus, one way to consider the importance of the Btk allosteric binding site for IP6is that it is needed to prevent down-regulation by competition of IP6for PIP3at the PIP3binding site. If true, Btk membrane binding may be an overstated facet of its regulation, as the authors also suggest. It will be fascinating in future studies to investigate selective mutants in vivo to dissect the relative contributions of PIP3and IP6in signaling.6 in regulating membrane-tethered Btk.This is an excellent point. We agree with reviewers’ comments and have expanded our discussion on the potential roles of IPAlternatively, just as IP6might stimulate dimerization of membrane-tethered Btk, as suggested in the Conclusions, might it not also enhance the affinity (avidity) of Btk for PIP3-containing membranes by promoting dimerization? This would have the effect of 'sensitizing' Btk to PIP3levels, and could be important. Such an effect of IP6(or IP7or IP8) could also/alternatively counteract inhibition of PH domain function by competition at the canonical site . It might thus selectively 'spare' Btk from such inhibition?We appreciate this point as well, and have included this in the concluding section.In the beginning of the Results and Discussion section, the authors describe the difference in the angles for the kinase SH3 domain with respect to other kinases and they explain the differences in orientation of the SH2 and SH3 domains, saying: “Coupling between the SH3 and SH2 domains may therefore be somewhat different in these kinases.” Although this may be true, it is also possible that there is not a fixed orientation in solution between the different domains and the differences reflect different arrangements captured by the crystals. The authors also say that: “diffuse electron density for the SH2 shows that this part of the polypeptide chain occupies a range of positions and adopts multiple conformations”. This statement seems to support such an idea.We agree with this comment.Still in the Results and Discussion section, the authors say that: “… nearly identical to those determined previously using NMR”. They should include RMSD for the C-alphas that were aligned.We thank the reviewers for pointing this out. We have calculated the RMSD value as suggested by the reviewers and added it to the Results section.This sentence, in the same section of the text: “The domain swap occurs in the same location within the SH2 domain as a similar swap seen in a Grb2 SH2-domain crystal structure.” From the drawing this similarity is not so apparent. They use the same helix against the same beta sheet, but the orientation of the two monomers appears different. In Grb2 the helices are essentially parallel to the beta-sheet whereas in Btk, these helices seem to be perpendicular.The relative orientation of the two subunits in the Btk SH2 dimer is rotated by about 90° with respect to that of the Grb2 SH2 dimer, as noted by the reviewers. We have deleted the word “similar” and replaced it by “domain”. The sentence now reads: “The domain swap occurs in the same location within the SH2 domain as a domain swap seen in a Grb2 SH2-domain crystal structure.”The authors say that it is unlikely “that a domain-swapped dimer” is physically important. That being the case, the authors may wish to consider reducing the overall discussion about domain swapping.We have considered this point. We have, however, decided to retain the discussion because the domain swap was an unexpected feature of the crystal structure.In the Results and Discussion, it says that “r.m.s. deviation from c-Src is 2.3 Å for the 24 α-carbons”. This seems to be a rather large deviation for so few carbons.The activation loop is a highly flexible part of the kinase domain. For example, when the active and inactive conformation of these 24 Cα atoms are compared between an active (PDB: 3LCK) and inactive (PDB: 1QCF) Src kinase conformation, the rms deviation is 11 Å. Thus 2.3 Å indicates that the conformations of the activation loops are similar.In that same section of the article, the authors call an Arg-Phe interaction an “amino-aromatic interaction”. Is this actually amino (cation)-aromatic pair, or is it a π-type (Pi-type) interaction between the guadinium of the Arg and the aromatic ring of the Phe?We have confirmed that “amino-aromatic” is a better description for these interactions in the crystal structure.In the subsection “Structure of a PH-TH-kinase construct of Btk”, it says: “The PH-TH module contacts the kinase N-lobe. The interface differs from the one found in Akt1”. Is this because they are intrinsically different or because of the deletion of the SH2 and SH3 domains? Some comment on this could be helpful.We thank the reviewers for pointing out this potential confusing statement in our manuscript. The PHkinase interface in Btk is very different from that in Akt1, and we have clarified this by adding a couple of sentences in the corresponding sections in the Results.Subsection headed “A structural model for full-length Btk”: it is not very clear what the cause is, but the deviations between the crystallographic structures and the ones at the end of the trajectories appear to be large. It is not very clear what these trajectories were probing and what the implications are for their having departed so significantly from the original structures?We thank the reviewers for pointing this out and we apologize that we did not make this clear in the manuscript. The relatively large deviation arises from the pivoting motions of the molecule during molecule dynamic simulations, and the use of only the C lobe of the kinase to align these structures, which amplified the deviations. The overall conformation of Btk at the end of the trajectories is actually very similar to the initial structure. We have now explained this in the Result section, and state that the rms deviation between the initial and final structures is only 2.3 Å when the alignment is done on the entire Src-like module.The manuscript says, in the “Activation of Btk by IP6in solution” section: “but these required higher protein concentration and longer times for appreciable autophosphylation than did IP6.” Looking atthe Coomassie-blue for some of the IP5doesn't show higher protein concentration.Perhaps the confusion here arises because the results of two different experiments (two different gels) are shown in Panel 5B. The top gel in this panel has lower protein concentration (2 μM Btk), shorter reaction time (5 minutes) and shorter exposure time. The bottom gel has higher Btk concentration (4 μM Btk), longer reaction time (10 minutes) and the gel has been exposed for a longer time. The figure legend has been modified to make this clearer.Subsection headed “The canonical lipid-binding pocket of the PH-TH module is not critical for IP6-mediated Btk activation”: when the authors note that R28C/N24E abolishes IP4binding and weakens IP6binding by 20-40-fold using ITC, they appear to conclude that the residual binding is solely due to the separate IP6site. Is it possible that the double mutant still allows for IP6binding to the IP4site? Perhaps the authors can clarify this section a bit more.6 to the IP4-binding deficient mutant is due to binding at the peripheral binding site. This is stated in the subsection “The peripheral binding site is critical for IP6-mediated Btk activation”.In ITC experiments, we saw no detectable binding when we added IP6 to a PH-TH module with mutations at both the canonical and the peripheral sites, which confirms that the residual binding of IPSubsection headed “Structure of the IP6-bound PH-TH module”: it is not said, although one can assume, that the dimers are two-fold symmetric. If that is the case it is not very clear whether or not the open-ended chain is a consequence of the crystal lattice rather than being biologically relevant. Therefore, the binding site that is formed by two molecules in the context of the open-ended chain of dimers may not be present in solution. The authors appear to imply that the IP6interaction site is mostly determined by one of the chains. If that were the case and IP6binds to that site even in the absence of the dimer-formed open-ended chain, then shouldn't one have observed a more complex stoichiometry in the ITC experiments (which shows an n∼1)? More explanation here could be helpful.We thank the reviewers for pointing out this potentially confusing point, which was not well addressed in the original manuscript. We agree that this chain of dimers is unlikely to be biologically relevant, and the dimer that matters for the Btk activation is the Saraste dimer, which we discuss extensively in the paper. We have now clarified this by reducing the discussion of the open-ended chain of dimers in the Results section, and adding several sentences to make it clear that the open-ended chain is likely to be only a feature of the crystals.The section on IP6regulation is fairly lengthy given the degree of speculation. The authors could consider condensing this.The molecular mechanism by which IP6 causes transient dimerization of Btk remains puzzling because we do not detect dimers even at concentrations as high as 30 μM. This section makes two points that we believe are important: (1) the conformation of the unliganded, monomeric Btk is likely to be different from that seen in the Saraste dimer and (2) changes in surface electrostatics can activate Btk in the absence of IP6. For these reasons we would prefer to retain this section.In the beginning of the Results and Discussion, it could be helpful to include a short introduction indicating which structures were determined as part of this work, to what resolution, and a reference toWe thank the reviewers for this suggestion. We have added a section in the beginning of the Results and Discussion summarizing our crystallographic work .It would be interesting to know whether the orientation of the IP6in the peripheral site is well defined or not. IP6binding to such a 'flat' (as the authors put it) site is often heterogeneous in pose, but the data insuggest that this might not be the case here, as does the second paragraph in “The peripheral binding site is critical for IP6-mediated Btk activation”. Some comment on stereospecificity would be helpful for those who think about IP6binding sites. Perhaps a density panel could be added toor its supplement?6 molecule can be determined unambiguously from the electron density because the phosphate group at position 2 juts out from the plane of the myo-inositol ring. This agrees well with our biochemical data that the phosphate at position 2 is likely to be critical for IP6 activation of Btk. We have added a figure showing electron density for IP6 at the peripheral site in 6 for this site in the Results section.We thank the reviewers for pointing out this important issue, which was not emphasized enough in the original manuscript. The disposition of the peripheral IPIn reflecting on the organization of the paper, one suggestion would be to place(“Structure of a PH-TH-kinase construct of Btk”) before. The story might flow/unfold better if the discovery of the potential autoinhibitory role of the PH-TH region leads into discovery of the Src-like disposition of domains in the Src-like module, then wrapped up nicely by the work inon whole Btk.We appreciate this point. We have found it to be challenging to weave all the threads of the discussion into one coherent narrative. At the end we find it easier to stay with the current organization.6 binding increases the on-rate of dimer formation of Btk by a factor of 10, and that once Btk is phosphorylated, the catalytic rate constant for transautophosphorylation increases by a factor of 10. Using physically reasonable values for the on and off rates and the catalytic rates we show that a small increase in on-rate for Btk dimer formation leads to a dramatic increase in the rate of autophosphorylation, even though the concentration of Btk is two orders of magnitude below the value of the dissociation constant. We think that this simple calculation helps explain an observation that might otherwise be counter-intuitive to some readers, which is that IP6 can activate Btk at Btk concentrations that are well below the dissociation constant for dimerization. Text referring to this new figure is in the last paragraph of “Fusing the Btk PH-TH module to the catalytic module of c-Abl promotes autophosphorylation of c-Abl in the prescence of IP6”.In addition to these changes made to the manuscript, in response to the reviewers’ comments, we have added one new figure . We used