Autoinhibitory Region Research Articles

AMP-activated protein kinase–mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β

The Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr144 phosphorylation by activated AMPK converts CaMKKβ into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKKβ.

Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase.

Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region.

SKIP controls lysosome positioning using a composite kinesin-1 heavy and light chain-binding domain.

ABSTRACTThe molecular interplay between cargo recognition and regulation of the activity of the kinesin-1 microtubule motor is not well understood. Using the lysosome adaptor SKIP (also known as PLEKHM2) as model cargo, we show that the kinesin heavy chains (KHCs), in addition to the kinesin light chains (KLCs), can recognize tryptophan-acidic-binding determinants on the cargo when presented in the context of an extended KHC-interacting domain. Mutational separation of KHC and KLC binding shows that both interactions are important for SKIP–kinesin-1 interaction in vitro and that KHC binding is important for lysosome transport in vivo. However, in the absence of KLCs, SKIP can only bind to KHC when autoinhibition is relieved, suggesting that the KLCs gate access to the KHCs. We propose a model whereby tryptophan-acidic cargo is first recognized by KLCs, resulting in destabilization of KHC autoinhibition. This primary event then makes accessible a second SKIP-binding site on the KHC C-terminal tail that is adjacent to the autoinhibitory IAK region. Thus, cargo recognition and concurrent activation of kinesin-1 proceed in hierarchical stepwise fashion driven by a dynamic network of inter- and intra-molecular interactions.

Structural Studies of IRF4 Reveal a Flexible Autoinhibitory Region and a Compact Linker Domain

IRF4 is a unique member of the interferon regulatory factor (IRF) family playing critical regulatory roles in immune cell development, regulation of obesity-induced inflammation, and control of thermogenic gene expression. The ability of IRF4 to control diverse transcriptional programs arises from its proficiency to interact with numerous transcriptional partners. In this study, we present the structural characterization of full-length IRF4. Using a combination of x-ray and small angle x-ray scattering studies, we reveal unique features of the interferon activation domain, including a set of β-sheets and loops that serve as the binding site for PU.1, and also show that unlike other IRF members, IRF4 has a flexible autoinhibitory region. In addition, we have determined the small angle x-ray scattering solution structure of full-length IRF4, which, together with circular dichroism studies, suggests that the linker region is not extended but folds into a domain structure.

Insight into the Mechanism of Intramolecular Inhibition of the Catalytic Activity of Sirtuin 2 (SIRT2).

Sirtuin 2 (SIRT2) is a NAD+-dependent deacetylase that has been associated with neurodegeneration and cancer. SIRT2 is composed of a central catalytic domain, the structure of which has been solved, and N- and C-terminal extensions that are thought to control SIRT2 function. However structural information of these N- and C-terminal regions is missing. Here, we provide the first full-length molecular models of SIRT2 in the absence and presence of NAD+. We also predict the structural alterations associated with phosphorylation of SIRT2 at S331, a modification that inhibits catalytic activity. Bioinformatics tools and molecular dynamics simulations, complemented by in vitro deacetylation assays, provide a consistent picture based on which the C-terminal region of SIRT2 is suggested to function as an autoinhibitory region. This has the capacity to partially occlude the NAD+ binding pocket or stabilize the NAD+ in a non-productive state. Furthermore, our simulations suggest that the phosphorylation at S331 causes large conformational changes in the C-terminal region that enhance the autoinhibitory activity, consistent with our previous findings that phosphorylation of S331 by cyclin-dependent kinases inhibits SIRT2 catalytic activity. The molecular insight into the role of the C-terminal region in controlling SIRT2 function described in this study may be useful for future design of selective inhibitors targeting SIRT2 for therapeutic applications.

Structural basis of Ets1 cooperative binding to palindromic sequences on stromelysin-1 promoter DNA.

Ets1 is a member of the Ets family of transcription factors. Ets1 is autoinhibited and its activation requires heterodimerization with a partner protein or DNA-mediated homodimerization for cooperative DNA binding. In the latter case, Ets1 molecules bind to palindromic sequences in which two Ets-binding sites (EBS) are separated by four base pairs, for example in the promoters of stromelysin-1 and p53. Interestingly, counteraction of autoinhibition requires the autoinhibitory region encoded by exon VII of the gene. The structural basis for the requirement of autoinhibitory sequences for Ets1 binding to palindromic EBS still remains unresolved. Here we report the crystal structure of two Ets1 molecules bound to an EBS palindrome of the stromelysin-1 promoter DNA, providing a plausible explanation for the requirement of exon VII-encoded sequences for Ets1 cooperative DNA binding. The proposed mechanism was verified both in vitro by surface plasmon resonance and in vivo by transcription-based assays.

Disorder in Protein Switches

Interactions of intrinsically disordered (ID) protein segments with partner proteins are often mediated by molecular recognition features (MoRFs). MoRFs in ID segments are not only used in interactions in trans but also in cis, i.e., they can mediate interactions between an ID segment and a domain that are both part of the same polypeptide chain. Such interactions in cis can switch the function of a protein domain off by steric or allosteric inhibition of substrate or binding partner access to catalytic sites or binding surfaces, respectively. This phenomenon is called autoinhibition. We demonstrate that most cis‐regulatory elements in proteins are ID and that the MoRFs in these ID autoinhibitory regions are highly conserved. We present results from molecular dynamics simulations that reveal the mechanism by which phosphorylation of MoRFs can relieve autoinhibition and introduce the first predictor that identifies ID autoinhibitory regions in proteins from sequence information only.

A New Cell-penetrating Peptide That Blocks the Autoinhibitory XIP Domain of NCX1 and Enhances Antiporter Activity

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na(+) ions for 1Ca(2+) ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIP(NCX1)), represent an autoinhibitory region involved in the Na(+)-dependent inactivation of the exchanger. Previous research has shown that an exogenous peptide having the same amino acid sequence as the XIP(NCX1) region exerts an inhibitory effect on NCX activity. In this study, we identified another regulatory peptide, named P1, which corresponds to the 562-688aa region of the exchanger. Patch-clamp analysis revealed that P1 increased the activity of the exchanger, whereas the XIP inhibited it. Furthermore, P1 colocalized with NCX1 thus suggesting a direct binding interaction. In addition, site-directed mutagenesis experiments revealed that the binding and the stimulatory effect of P1 requires a functional XIP(NCX1) domain on NCX1 thereby suggesting that P1 increases the exchanger activity by counteracting the action of this autoinhibitory sequence. Taken together, these results open a new strategy for developing peptidomimetic compounds that, by mimicking the functional pharmacophore of P1, might increase NCX1 activity and thus exert a therapeutic action in those diseases in which an increase in NCX1 activity might be helpful.

Receptor kinase-mediated control of primary active proton pumping at the plasma membrane.

Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2 and PSY1R observed might provide a general paradigm for regulation of plasma membrane proton transport by receptor kinases.

Mutation of Vav1 adaptor region reveals a new oncogenic activation.

Vav family members function as remarkable scaffold proteins that exhibit both GDP/GTP exchange activity for Rho/Rac GTPases and numerous protein-protein interactions via three adaptor Src-homology domains. The exchange activity is under the unique regulation by phosphorylation of tyrosine residues hidden by intra-molecular interactions. Deletion of the autoinhibitory N-terminal region results in an oncogenic protein, onco-Vav, leading to a potent activation of Rac GTPases whereas the proto-oncogene barely leads to transformation. Substitution of conserved residues of the SH2-SH3 adaptor region in onco-Vav reverses oncogenicity. While a unique substitution D797N did not affect transformation induced by onco-Vav, we demonstrate that this single substitution leads to transformation in the Vav1 proto-oncogene highlighting the pivotal role of the adaptor region. Moreover, we identified the cell junction protein β-catenin as a new Vav1 interacting partner. We show that the oncogenicity of activated Vav1 proto-oncogene is associated with a non-degradative phosphorylation of β-catenin at residues important for its functions and its redistribution along the cell membrane in fibroblasts. In addition, a similar interaction is evidenced in epithelial lung cancer cells expressing ectopically Vav1. In these cells, Vav1 is also involved in the modulation of β-catenin phosphorylation. Altogether, our data highlight that only a single mutation in the proto-oncogene Vav1 enhances tumorigenicity.

Abstract 2956: The role of apoptotic blebbing in tissue homeostasis and cancer

Abstract Efficient apoptotic cell clearance prevents the release of potentially immunogenic intracellular contents into the surrounding environment to maintain tissue homeostasis; however, the importance of membrane blebbing for apoptotic cell recognition and clearance has not been established. To address fundamental questions regarding the influence of ROCK1 cleavage and consequent apoptotic membrane blebbing in cellular corpse clearance and the maintenance of tissue homeostasis, mice were generated with a single amino acid substitution in the caspase cleavage site (D1113A) that converts ROCK1 to a non-cleavable (ROCK1nc) form. We previously showed that during apoptosis, caspase cleavage of ROCK1 removes an auto-inhibitory carboxy-terminal region to yield a constitutively active kinase fragment that leads to phosphorylation of downstream targets which promote contractile force generation leading to shrinkage, blebbing and nuclear disintegration. When apoptosis was induced in mouse embryonic fibroblasts, the biochemical apoptotic programme itself was unaffected, although cells had significantly impaired morphological alterations. Furthermore, by imaging apoptosis of GFP labelled melanoblasts in ex vivo embryonic skin explants, the effect of the ROCK1nc point mutation was to impair typical apoptotic morphological changes. To understand the biological role of membrane blebbing in the maintenance of tissue homeostasis, we used the liver-selective genotoxic compound diethylnitrosamine (DEN) to induce acute tissue damage. Following DEN treatment, we found that TUNEL positive apoptotic cells were inefficiently cleared in ROCK1nc mice, which resulted in increased neutrophil infiltration in response to the accumulating apoptotic debris. Histologically, the ROCK1nc livers appeared more damaged with evidence of steatohepatitis and perivenular damage, which was accompanied by higher serum alanine transferase levels. We then asked whether defects in tissue damage responses in ROCK1nc mice would affect tumor development in a model of DEN-induced hepatocellular carcinoma. Interestingly, ROCK1nc mice had reduced tumor volume and numbers, suggesting that defective blebbing may be beneficial for reducing tumor initiation and growth. Taken together, our results indicate that efficiency of phagocytic clearance may contribute to cancer susceptibility. Citation Format: Linda Julian, Michael F. Olson. The role of apoptotic blebbing in tissue homeostasis and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2014-2956

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47(phox) is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47(phox) phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47(phox) protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47(phox) is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22(phox) binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47(phox-/-) coronary microvascular cells. Compared with wild-type p47(phox) cDNA transfected cells, the single mutation of S379A completely blocked p47(phox) membrane translocation, binding to p22(phox) and endothelial O2(·-) production in response to acute stimulation of PKC. p47(phox) C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47(phox) conformational changes and NADPH oxidase-dependent superoxide production by cells.

The auto-inhibitory domain and ATP-independent microtubulebinding region of Kinesin heavy chain are major functional domains for transport in the Drosophila germline

The auto-inhibitory domain and ATP-independent microtubulebinding region of Kinesin heavy chain are major functional domains for transport in the <i>Drosophila</i> germline

Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under invitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

An Ire1–Phk1 Chimera Reveals a Dispensable Role of Autokinase Activity in Endoplasmic Reticulum Stress Response

The endoplasmic reticulum transmembrane receptor Ire1 senses over-accumulation of unfolded proteins in the endoplasmic reticulum and initiates the unfolded protein response (UPR). The cytoplasmic portion of Ire1 has a protein kinase domain (KD) and a kinase extension nuclease (KEN) domain that cleaves an mRNA for encoding the Hac1 transcription factor needed to express UPR genes. During this UPR signaling, Ire1 proteins self-assemble into an oligomer of dimers, which essentially requires autophosphorylation of a constituent activation loop in the KD. However, it is not clear how dimerization, autophosphorylation, and KEN domain function are precisely coordinated. In this study, we uncoupled the KD and KEN domain functions, by removing the activation loop along with an extended region that we called the auto-inhibitory region (AIR), or by swapping the activation loop with a homologous loop from phosphorylase kinase 1 (Ire1PHK). Both Ire1ΔAIR and Ire1PHK activated the UPR even when either protein contained a mutation (D797A) that abolished the ability of Ire1 KD to transfer phosphates to the AIR. Neither protein functioned when containing mutations in key ATP binding residues (E746A and N749A) or in residues that disrupted Ire1 dimer interface (W426A or R697D). We interpret these results as evidence supporting the notion that the primary function of the kinase domain is to autophosphorylate the AIR in order to relieve auto-inhibition and that ADP acts as a switch to activate the KEN domain-catalyzed HAC1 mRNA cleavage.

Mapping the Location of Autoinhibitory B-Domain Motifs within Factor V: New Insights Into How Factor V Activation Unfolds

Abstract 495Blood coagulation factor V (FV) circulates as a procofactor with little or no procoagulant activity. Proteolytic removal of a large central B-domain converts FV (domain organization of A1-A2-B-A3-C1-C2) to an active cofactor for membrane-bound FXa to form prothrombinase. Recently we found that discrete, evolutionarily conserved sequences within the B-domain serve an autoinhibitory function and are necessary to maintain FV as an inactive procofactor. The autoinhibitory region within the B-domain (termed the procofactor regulatory region or PRR) is sequence specific and remarkably short (∼100 amino acids out of 836 from B-domain) consisting of basic (963–1008; basic region or BR) and acidic (1493–1537; acidic region or AR) sequences. Dismantling this region appears to be the driving force to unveil a high affinity binding site(s) for FXa and thus underpins the procofactor to cofactor transition. While the BR and AR seem to work together to provide “on-site” repression of cofactor activity, there is no structural information about the FV B-domain that could provide clues into how this critical region functions. Here we use B-domain fragments tethered to an artificial protease in order to map the position of the PRR relative to the rest of the FV molecule. A BR peptide with an introduced free Cys at position 990 was stoichiometrically modified with FeBABE (Fe-p-bromoacetamidobenzyl-EDTA). FeBABE is a protein cutting reagent with a sulfhydryl-reactive moiety for attachment to Cys and an EDTA-chelated iron atom which, when triggered with ascorbic acid and peroxide, generates hydroxyl radicals that cut peptide bonds in a sequence-independent fashion. When a protein labeled with FeBABE binds its target, the Fe-BABE moiety facilitates cutting if it is orientated correctly and near (<12 Å) the contact site of the target protein. In our experimental system, we employed a constitutively active B-domainless form of FV (FV-810) that harbors the AR but lacks the BR. Initial studies revealed that the BR peptide, without or tethered with FeBABE, binds to FV-810 with high affinity (nM) and inhibits cofactor activity. Proteolysis of FV-810 by BR-FeBABE, was visualized by Western blot, using monoclonal antibodies recognizing either the heavy or light chains. Using this approach, we consistently found that BR-FeBABE cut FV-810 predominantly at two sites. The first is at the C-terminal end of the B-domain. This region is enriched in acidic residues and represents the AR defined above. The second site of cleavage is within the heavy chain. While we cannot exclude the possibility that the site of contact is at the N-terminus of the A1 domain, based on the proposed 3D structure of FVa, it is more likely that the BR is binding near the C-terminal end of the A2 domain which is also enriched in acidic amino acids. Compared to a panel of FVa derivatives with variably truncated heavy chains, we suggest that BR-FeBABE cuts FV-810 near residues 675–685. Overall the data suggest that the B-domain must be folded in such a way as to place the BR in close spatial proximity to the AR at the C-terminal end of the B-domain and also to the C-terminal heavy chain region. Consistent with the idea that these interacting motifs work to keep FV as a procofactor by suppressing FXa binding, adding saturating amounts of FXa, but not the zymogen FX, prevented the BR-FeBABE induced cleavage of FV-810. Similarly, activated protein C (APC) which is thought to share a related binding site with FXa, also prevented BR-FeBABE from cutting FV-810. Addition of excess unlabeled BR peptide substantially reduced cutting of FV-810 by BR-FeBABE, indicating that proteolysis is not due to non-specific cleavage. Additionally, removing the basic charge of BR-FeBABE via acetylation eliminated binding to FV-810 and also any signs of specific cutting. Further, FV-derivatives harboring both the AR and BR in cis and hence not binding the BR peptide, were not cut by excess BR-FeBABE added in trans. In conclusion, these findings provide new insights into the spatial relationship between key regulatory B-domain motifs and the core heavy/light chain region. These interacting autoinhibitory motifs directly or indirectly obscure FXa binding and hence stabilize the inactive procofactor state of FV. These studies also document a remarkably powerful way to map the spatial relationship between regions of proteins and provide new information on coagulation factor binding sites. Disclosures:Camire:Pfizer: Patents & Royalties, Research Funding.

A Bipartite Autoinhibitory Region within the B-domain Suppresses Function in Factor V

Activation of blood coagulation factor V (FV) is a key reaction of hemostasis. FV circulates in plasma as an inactive procofactor, and proteolytic removal of a large central B-domain converts it to an active cofactor (FVa) for factor Xa (FXa). Here we show that two short evolutionary conserved segments of the B-domain, together termed the procofactor regulatory region, serve an essential autoinhibitory function. This newly identified motif consists of a basic (963-1008) and an acidic (1493-1537) region and defines the minimal sequence requirements to maintain FV as a procofactor. Our data suggest that dismantling this autoinhibitory region via deletion or proteolysis is the driving force to unveil a high affinity binding site(s) for FXa. These findings document an unexpected sequence-specific role for the B-domain by negatively regulating FV function and preventing activity of the procofactor. These new mechanistic insights point to new ways in which the FV procofactor to cofactor transition could be modulated to alter hemostasis.

Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker

Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.

Pannexin 1, an ATP Release Channel, Is Activated by Caspase Cleavage of Its Pore-associated C-terminal Autoinhibitory Region

Pannexin 1 (PANX1) channels mediate release of ATP, a "find-me" signal that recruits macrophages to apoptotic cells; PANX1 activation during apoptosis requires caspase-mediated cleavage of PANX1 at its C terminus, but how the C terminus inhibits basal channel activity is not understood. Here, we provide evidence suggesting that the C terminus interacts with the human PANX1 (hPANX1) pore and that cleavage-mediated channel activation requires disruption of this inhibitory interaction. Basally silent hPANX1 channels localized on the cell membrane could be activated directly by protease-mediated C-terminal cleavage, without additional apoptotic effectors. By serial deletion, we identified a C-terminal region just distal to the caspase cleavage site that is required for inhibition of hPANX1; point mutations within this small region resulted in partial activation of full-length hPANX1. Consistent with the C-terminal tail functioning as a pore blocker, we found that truncated and constitutively active hPANX1 channels could be inhibited, in trans, by the isolated hPANX1 C terminus either in cells or when applied directly as a purified peptide in inside-out patch recordings. Furthermore, using a cysteine cross-linking approach, we showed that relief of inhibition following cleavage requires dissociation of the C terminus from the channel pore. Collectively, these data suggest a mechanism of hPANX1 channel regulation whereby the intact, pore-associated C terminus inhibits the full-length hPANX1 channel and a remarkably well placed caspase cleavage site allows effective removal of key inhibitory C-terminal determinants to activate hPANX1.

V559A and N822I double KIT mutant melanoma with predictable response to imatinib?

Dear Editor, Since the initial report of KIT mutations in subtypes of melanoma, several reports of KIT-targeted therapies have been published (Carvajal et al., 2009; Hodi et al., 2008; Lutzky et al., 2008). Activating KIT mutations have been documented in a variety of neoplastic diseases including, in addition to melanoma, mastocystosis, acute myeloid leukemia, seminoma and gastrointestinal stromal tumors (Went et al., 2004). Herein, we report a case of a KIT double mutation in anorectal melanoma not previously described in melanoma, but with a response to imatinib mesylate that might have been expected given the knowledge from other cancers. The patient is an 87-year-old woman who developed constipation and a palpable rectal mass in the summer of 2009. Biopsy of the mass demonstrated an ulcerated melanoma invading the submucosa and muscle to a depth of approximately 11 mm with angiolymphatic invasion. Computerized tomography (CT) scans of the chest, abdomen and pelvis revealed metastatic disease in the pelvis, inguinal region and lungs. Substantial growth of the disease was documented on serial CT scans 3 months apart with the largest lung nodule increasing 44% (14 to 25 mm). The patient declined treatment with systemic chemotherapy and was not a candidate for the available clinical trials but she did express an interest in some form of therapy. Her melanoma was assessed for KIT mutational status. A double mutation was identified, comprising the exon 11 mutation V559A and the exon 17 mutation N822I. The patient subsequently initiated treatment with imatinib 400 mg daily in November 2009. Surveillance CT scans in February 2010 revealed that most metastatic lesions were stable to minimally decreased, including a right inguinal lymph node (16 to 14 mm), a right upper lobe lung nodule (20 to 18 mm), a right lower lobe lung nodule (25 to 23 mm) and a right ischioanal lesion (19 to 15 mm). There was complete disappearance of multiple subcentimetre lung nodules; however, there was also some growth of other lung nodules, the largest of which increased from 25 to 28 mm. The findings were consistent with a mixed response, although in accordance with RECIST criteria, the patient’s response would best be classified as stable disease ( 7% decrease in sum of diameters of target lesions). No repeat biopsy was attempted. The dose of imatinib was titrated from 400 to 800 mg with the aim of overcoming any possible resistance to therapy and improving the patient’s response. Unfortunately, the patient developed significant fatigue, dyspnea on exertion and lower extremity edema. Treatment was held, and after recovery from these acute symptoms, attempts were made to reinitiate imatinib at the lower dose of 400 mg. The patient was not able to tolerate resumption of therapy, and she experienced further clinical decline and progression of disease; ultimately, she succumbed to her metastatic melanoma in August, 2010. Efforts to define the genetic landscape of melanomas have led to the recognition that alterations in the KIT gene constitute the predominant mutation in melanomas arising from chronic sun-damaged skin and acral and mucosal anatomical locations (Curtin et al., 2005, 2006). KIT mutations in melanoma are now only beginning to be catalogued. The experience with KIT mutations in GISTs, however, is much more extensive, and this experience provides a conceptual framework for understanding the pathobiology of and therapeutic approaches to treating neoplastic diseases driven by KIT mutations (Corless and Heinrich, 2008). KIT mutations underlie approximately 85% of GISTs; and the vast majority ( 70%) occurs within exon 11 corresponding to the autoinhibitory juxtamembrane domain. KIT exon 9 lies within the extracellular ligand-binding region, and mutations within this exon account for 10 to 15% of KIT mutations in GIST. KIT mutations within the kinase domain (either exon 13 or exon 17) occur with much less frequency (<5%). To date, molecular analyses have identified approximately two dozen unique KIT mutations in melanoma (Woodman and Davies, 2010). Similar to GIST, the majority of KIT mutations in melanoma occur within the juxtamembrane autoinhibitory region encoded by exon 11. Unique to melanoma, though, the