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Protein Kinase C and Guanosine Triphosphate Combine to Potentiate Calcium-dependent Membrane Fusion Driven by Annexin 7*

  • Hung Caohuy
    Affiliations
    From the Department of Anatomy, Physiology, and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
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  • Harvey B. Pollard
    Correspondence
    To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, Uniformed Services University School of Medicine, USUHS, Bethesda, MD 20814. Tel.: 301-295-3200; Fax: 301-295-1715;
    Affiliations
    From the Department of Anatomy, Physiology, and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:May 06, 2002DOI:https://doi.org/10.1074/jbc.M202452200
      Exocytotic secretion is promoted by the concerted action of calcium, guanine nucleotide, and protein kinase C. We now show that the calcium-dependent membrane fusion activity of annexin 7 in vitro is further potentiated by the combined addition of guanine nucleotide and protein kinase C. The observed increment involves the simultaneous activation of annexin 7 by these two effectors. Guanosine triphosphate (GTP) and its non-hydrolyzable analogues optimally enhance the phosphorylation of annexin 7 by protein kinase C in vitro. Reciprocally, phosphorylation by protein kinase C significantly potentiates the binding and hydrolysis of GTP by annexin 7. Only protein kinase C-dependent phosphorylation has a significant positive effect on annexin 7 GTPase, although other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and pp60c- src , have been shown to label the protein with high efficiency. In vivo, the ratio of bound GDP/GTP and phosphorylation of annexin 7 change in direct proportion to the extent of catecholamine release from chromaffin cells in response to stimulation by carbachol, or to inhibition by various protein kinase C inhibitors. These results thus lead us to hypothesize that annexin 7 may serve as a common site of action for calcium, guanine nucleotide, and protein kinase C in the exocytotic membrane fusion process in chromaffin cells.
      GTP
      guanosine 5′-triphosphate
      ANX7
      annexin 7
      cGMP
      cyclic guanosine monophosphate
      GDPβS
      guanosine 5′-O-(2-thiodiphosphate)
      GMP-P(NH)P
      guanyl-5′-yl imidodiphosphate
      GTPγS
      guanosine 5′-O-(3-thiotriphosphate)
      MES
      2-(N-morpholino)ethanesulfonic acid
      PKC
      protein kinase C
      PS
      phosphatidylserine
      PIPES
      piperazine-N,N′-bis(2-ethanesulfonic acid)
      PKAcat
      catalytic subunit of cAMP-dependent protein kinase
      PKG
      cGMP-dependent protein kinase
      PMA
      phorbol 12-myristate 13-acetate
      SNARE
      soluble NSF attachment protein receptors
      Guanosine triphosphate (GTP)1 and its non-hydrolyzable analogues (i.e. GTPγS and GMP-P(NH)P) are known to promote Ca2+-dependent exocytotic secretion from chromaffin cells and many other cell types (
      • Barrowman M.M.
      • Cockcroft S.
      • Gomperts B.D.
      ,
      • Howell T.W.
      • Cockcroft S.
      • Gomperts B.D.
      ,
      • Haslam R.J.
      • Davidson M.M.L.
      ,
      • Luini A.
      • DeMatteis M.A.
      ,
      • Vallar L.
      • Biden T.J.
      • Wollheim C.B.
      ,
      • Oetting M.
      • LeBoff M.
      • Swiston L.
      • Preston J.
      • Brown E.
      ,
      • Knight D.E.
      • Baker M.F.
      ,
      • Bittner M.A.
      • Holz R.W.
      • Neubig R.R.
      ,
      • Bader M.F.
      • Sontag J.M.
      • Thierse D.
      • Aunis D.
      ,
      • Ahner-Hilger G.
      • Wegenhorst U.
      • Stecher B.
      • Spicher K.
      • Rosenthal W.
      • Gartzl M.
      ). Likewise, activation of protein kinase C (PKC) has been shown to trigger Ca2+-dependent secretion in these secreting cell types as well (11–24). Furthermore, many permeabilized cell studies have also supported a role for PKC in further enhancing the stimulatory action of Ca2+ and GTP in the exocytotic process (
      • Bader M.F.
      • Sontag J.M.
      • Thierse D.
      • Aunis D.
      ,
      • Smolen J.E.
      • Sandborg R.R.
      ,
      • Sloan D.C.
      • Haslam R.J.
      ,
      • Cockcroft S.
      • Howell T.W.
      • Gomperts B.D.
      ,
      • Ahnert-Hilger G.
      • Brautigam M.
      • Gratzl M.
      ,
      • Lillie T.H.
      • Gomperts B.D.
      ,
      • Koopmann W.R.
      • Jackson R.C.
      ,
      • Carroll A.G.
      • Rhoads A.R.
      • Wagner P.D.
      ). These observations thus have led to the proposal of a hypothetical exocytotic model by Lillie and Gomperts (
      • Lillie T.H.W.
      • Gomperts B.D.
      ) in which Ca2+, GTP, and PKC act in concert in a regulatory sequence leading to exocytosis (
      • Lillie T.H.W.
      • Gomperts B.D.
      ). In this model, two GTP-binding proteins are involved in controlling the stimulus-secretion process. The first GTP-binding protein is the putative receptor-linked G-protein (GP) that controls the activity of phospholipase C, thereby generating inositol 1,4,5-trisphosphate and diacylglycerol. Further downstream from the signal transduction level, a second GTP-binding protein (G E , E for exocytosis), a putative GTPase so far undefined as a molecular entity, acts in parallel, or might be closely associated with a Ca2+-binding protein at the docking/fusion site of the exocytotic machinery. Subsequent phosphorylation by diacylglycerol-activated PKC triggers these proteins into mediating the exocytotic membrane fusion process. Although phenomenologically well known, the specific sites of action of Ca2+, GTP, and PKC in the stimulus-secretion cascade remain unknown.
      Annexin 7 (ANX7; synexin) is a Ca2+-dependent membrane fusion protein (
      • Creutz C.E.
      • Scott J.H.
      • Pazoles C.J.
      • Pollard H.B.
      ,
      • Hong K.
      • Duzgunes N.
      • Ekerdt R.
      • Papahadjopoulos D.
      ,
      • Nir S.
      • Stutzin A.
      • Pollard H.B.
      ,
      • Pollard H.B.
      • Rojas E.
      • Burns A.L.
      ), for which recent evidence has strongly suggested the possibility of its involvement in exocytosis. For example, we have reported that ANX7 is a Ca2+-activated GTPase, both in vitro and in secreting chromaffin cells, and that in vitro membrane fusion activity of ANX7 is further enhanced upon binding to GTP (
      • Caohuy H.
      • Srivastava M.
      • Pollard H.B.
      ). More recently, we have reported that the heterozygous knockout Anx7(+/−) mouse suffers from an insulin secretion deficit from islets of Langerhans, as well as defective Ca2+ signaling processes in β-cells (
      • Srivastava M.
      • Atwater I.
      • Glasman M.
      • Leighton X.
      • Goping G.
      • Caohuy H.
      • Miller G.
      • Pichel J.
      • Wesphal H.
      • Mears D.
      • Rojas E.
      • Pollard H.B.
      ). Furthermore, we have reported that ANX7 is phosphorylated by PKC, both in vitro and in secreting chromaffin cells (
      • Caohuy H.
      • Pollard H.B.
      ). Phosphorylation by PKC significantly potentiates the ability of ANX7 to fuse phospholipid vesicles, and the apparent K 12 of Ca2+is lowered from 200 to 50 μm (
      • Caohuy H.
      • Pollard H.B.
      ). Sequence and site-directed mutagenesis studies of ANX7 have shown that putative binding sites for GTP are located in proximity to consensus phosphorylation sites for PKC. These data thus have led us to hypothesize that these two processes may modulate the action of each other in activating ANX7-driven membrane fusion. To test this hypothesis, we have investigated the interconnections between PKC and GTP action on the Ca2+ dependence of ANX7-driven membrane fusion both in vitro and in vivo.
      In this study, we report that GTPγS and PKC both mutually enhance the binding of each other to ANX7, and also potentiate Ca2+-dependent membrane fusion driven by ANX7.In vitro, phosphorylation of ANX7 by PKC is optimally enhanced by GTP and its non-hydrolyzable analogues. Reciprocally, the binding and hydrolysis of GTP by ANX7 are markedly potentiated by PKC-catalyzed phosphorylation. Whereas certain other kinases label ANX7 efficiently, they do not substitute for PKC in potentiating GTP binding or membrane fusion. In vivo, we find that for ANX7, both the ratio of bound GDP/GTP as well as phosphorylation by PKC change in proportion to the extent of catecholamine release from stimulated chromaffin cells. Thus, GTP and PKC combine specifically to transform ANX7 into a highly efficient Ca2+-dependent membrane fusogen. We therefore conclude that the membrane fusion machinery might include ANX7 as a common site of action for Ca2+, GTP, and PKC in the exocytotic membrane fusion process.

      DISCUSSION

      GTP and PKC, in concert with Ca2+, are known to constitute a highly potent intracellular effector system for exocytosis in a variety of secreting cell types (1–29). In addition, Lillie and Gomperts (
      • Lillie T.H.W.
      • Gomperts B.D.
      ) have suggested that these effectors may exert their positive actions either directly on a common site, or on putative target proteins that are closely associated with each other in the exocytotic machinery. Based on our previous studies (
      • Caohuy H.
      • Srivastava M.
      • Pollard H.B.
      ,
      • Caohuy H.
      • Pollard H.B.
      ), we have proposed that ANX7 might function as a common site for these effectors in the exocytotic machinery. To further support this hypothesis, we demonstrate here that the Ca2+-dependent lipid vesicle fusion activity of ANX7 in vitro is significantly amplified by the combination of GTPγS and PKC (Fig. 1). Furthermore, the in vitro data on ANX7 membrane fusion activity appear to be well correlated with what we have observed in vivo. In these in vivo studies with ANX7, the ratio of bound GDP/GTP and phosphorylation by PKC change in direct proportion to the extent of catecholamine release from 33P-labeled chromaffin cells in response to stimulation by carbachol, or to inhibition by various PKC inhibitors. This close correlation between the in vivo andin vitro data implies that ANX7 functionally behaves like a G E (G-protein for exocytosis; Ref.
      • Lillie T.H.W.
      • Gomperts B.D.
      ), and transduces the intracellular signals for exocytosis by simultaneously binding GTP and being phosphorylated by PKC in a Ca2+-dependent manner.
      Based on these findings, the simplest explanation for the observed additive effects of PKC and GTPγS on ANX7 membrane fusion activityin vitro involves the same mechanisms of activation induced by these two agents in vitro. Indeed, our further in vitro analyses have shown that the combined presence of guanine nucleotides and PKC in the reaction mixture simultaneously increases the sensitivity to the action of each other on ANX7.

      Guanine Nucleotides Stimulate ANX7 Phosphorylation by PKC

      With regard to the PKC action on ANX7, we have found that the efficiency of ANX7 phosphorylation by PKC in vitro is further enhanced by GTP and its non-hydrolyzable analogues, but not by GDPβS (Fig. 2). Significantly, the concentrations of added guanine nucleotides that activate this ANX7 phosphorylation event are relevant to the physiological GTP concentration range (
      • Otero A.D.
      ,
      • Bourne H.R.
      • Sanders D.A.
      • McCormick F.
      ). These data clearly imply that the binding of GTP and its non-hydrolyzable analogues to ANX7 can confer conformational flexibility that makes ANX7 phosphorylation sites more accessible to PKC. Because ANX7 is a Ca2+-dependent GTPase (
      • Caohuy H.
      • Srivastava M.
      • Pollard H.B.
      ), this implication appears to be relevant because increased conformational flexibility of GTP binding is a common feature of most GTPases. Such flexibility enables GTPase proteins to function as molecular switches in which GTP- and GDP-bound forms have different conformations, and therefore significantly different activities (
      • Melancon P.
      ). To further support this concept, we have found that activation of ANX7 phosphorylation by guanine nucleotides is not attributed to changes in PKC activity. The evidence is that the levels of autophosphorylation of PKC, which are proportional to the activity of the kinase (
      • Newton A.C.
      ), remain relatively constant under all experimental conditions tested (Fig. 2, Aand C). Furthermore, we have found that the rank order of effectiveness for ANX7 phosphorylation by PKC is GTPγS > GMP-P(NH)P > GTP ≫ GDPβS (Fig. 2 A). This finding indicates that the binding of GTP rather than its hydrolysis is of critical importance for the phosphorylation process. In addition, the finding indicates that this modification is specifically sensitive to the activated, GTP-bound form of ANX7. Together with the data from our previously published study (
      • Caohuy H.
      • Pollard H.B.
      ), the present data strongly suggest that GTP further enhances the synergistic action of the elevated Ca2+ concentration and the slightly acidic pH (i.e. pH 6.8) in transforming ANX7 into a highly susceptible substrate for phosphorylation by PKC. Significantly, this optimal condition for the in vitro ANX7 phosphorylation by PKC appears to be physiologically relevant, because all of these factors are observed to be localized endogenously, as in the case of GTP, or to change coincidently, as in the case of Ca2+ concentration and pH, in the cell during stimulation.

      Phosphorylation by PKC Stimulates the Ability of ANX7 to Bind and Hydrolyze GTP

      Reciprocally, the ANX7 phosphorylation by PKC substantially stimulates the basal levels of GTP binding and GTP hydrolysis by ANX7 (Figs. Figure 3, Figure 4, Figure 5). The significance of these results is that upon phosphorylation by PKC, the turnover number for ANX7 is now relatively equivalent to those of some known G-proteins, including EF-G, EF-Tu, tubulin, and the G components of adenylate cyclase and transducin, with turnover numbers of 0.012–0.25 mol/min/mol of protein (
      • Chinali G.
      • Parmeggiania A.
      ,
      • Fasano O.
      • DeVenditis E.
      • Parmeggiani A.
      ,
      • Carlier M.-F.
      • Pantaloni D.
      ,
      • Brandt D.R.
      ,
      • Fung B.K.-K.
      ). Furthermore, the rate of GTP binding for phosphorylated ANX7 is quite similar to that of GTP hydrolysis, indicating that the hydrolytic/exchange reaction is rapid and is limited by GDP dissociation. Such an indication is supported by the present data (Fig.1 B) showing that the addition of excess GDPβS markedly inhibits the additive effect of PKC and GTPγS on ANX7 membrane fusion activity. This result strongly suggests that the exchange reaction of GDPβS for GTPγS is blocked by the excess molar concentration of GDPβS. Thus, it appears from these data that once ANX7-bound GTP is hydrolyzed, the newly formed GDP is released rapidly, and the empty nucleotide-binding pocket of ANX7 is ready to accommodate a new GTP molecule. ANX7 has a higher affinity for GTP than it has for GDP, thus indicating that the exchange is supported by energetic properties (
      • Caohuy H.
      • Srivastava M.
      • Pollard H.B.
      ).
      At present, the mechanisms by which both GTP binding and GTP hydrolysis by ANX7 are enhanced by PKC phosphorylation remain to be fully elucidated. Nonetheless, it is plausible to speculate that the ANX7 conformational change induced by the PKC phosphorylation event is instrumental for both the stimulated GTPase activity and the rapid GDP/GTP exchange.

      No Effects of Phosphorylation by Other Protein Kinases on ANX7 GTPase Activity

      The in vitro studies have shown that, in a simultaneous GTPase-phosphorylation reaction, PKA-, PKG-, and pp60c- src -catalyzed phosphorylation do not significantly alter the molar turnover number of the GTPase reaction mediated by ANX7 (Fig. 6). These results strongly suggest that, unlike PKC, these kinases may phosphorylate ANX7 on sites distant to the GTP-binding site, and are incapable of influencing the binding and hydrolysis of GTP of ANX7. Thus, the lack of stimulation by PKA, PKG, and pp60c- src on both the guanine nucleotide binding/hydrolysis property and on the membrane fusion activity (Fig.1 C) of ANX7 coincides with other observations showing that these kinases are not directly involved in regulated exocytosis (
      • Bittner M.A.
      • Holz R.W.
      • Neubig R.R.
      ,
      • Cheek T.R.
      • Burgoyne R.D.
      ,
      • Shono M.
      • Houchi H.
      • Oka M.
      • Nakaya Y.
      ,
      • Kumai T.
      • Tanaka M.
      • Tateishi T.
      • Asoh M.
      • Kobayashi S.
      ,
      • Ely C.M.
      • Oddie K.M.
      • Litz J.S.
      • Rossomando A.J.
      • Kanner S.B.
      • Sturgill T.W.
      • Parson S.J.
      ,
      • Ohnishi H.
      • Yamamori S.
      • Ono K.
      • Aoyagi K.
      • Kondo S.
      • Takahashi M.
      ). In addition, the lack of effects of these phosphorylation events, compared with the consequences of PKC, serves as an important control for emphasizing the importance of PKC-induced changes in ANX7 function.

      Membrane Fusion Cycle of ANX7

      Based on all of the present observations and of those in previously published reports (
      • Caohuy H.
      • Srivastava M.
      • Pollard H.B.
      ,
      • Caohuy H.
      • Pollard H.B.
      ), we propose the following ANX7-driven membrane fusion cycle (see Fig.8). Under a resting, low-Ca2+condition, ANX7 exists in an inactive state (ANX7-Mg2+-GDP), which is formed by a process of constitutive Mg2+-dependent hydrolysis of GTP (Fig. 8, (
      • Barrowman M.M.
      • Cockcroft S.
      • Gomperts B.D.
      )). Upon elevation of Ca2+, ANX7 binds Ca2+, transforming into a moderately active form (ANX7-Ca2+/Mg2+-GDP), which can drive membrane fusion (Fig. 8, (
      • Howell T.W.
      • Cockcroft S.
      • Gomperts B.D.
      )). GDP bound to this form can be replaced by GTP, and in this (ANX7-Ca2+/Mg2+-GTP) form, membrane fusion activity of ANX7 is further activated (Fig. 8, (
      • Haslam R.J.
      • Davidson M.M.L.
      )). Upon hydrolysis of GTP to GDP, a transient (ANX7-Ca2+/Mg2+-GDP) complex is formed (Fig. 8, (
      • Luini A.
      • DeMatteis M.A.
      ,
      • Vallar L.
      • Biden T.J.
      • Wollheim C.B.
      )). Under a suitable phosphorylation condition, either the GDP- or the GTP-bound forms of ANX7 can be phosphorylated by PKC, and PKC phosphorylation oscillates ANX7 between two phosphorylated states, (P-(ANX7-Ca2+/Mg2+-GTP)) and (P-(ANX7-Ca2+/Mg2+-GDP)), by stimulating the intrinsic GTPase and GTP/GDP exchange activities of ANX7. As a result, the membrane fusion activity of ANX7, with an order of efficiency (P-(ANX7-Ca2+/Mg2+-GTP)) > (P-(ANX7-Ca2+/Mg2+-GDP)), is at the optimal level, even operating at lower Ca2+ concentrations (Fig. 8, (
      • Oetting M.
      • LeBoff M.
      • Swiston L.
      • Preston J.
      • Brown E.
      )). The GDP-bound, phosphorylated ANX7 is subsequently dephosphorylated by the action of a serine/threonine-protein phosphatase which, we have now learned, is calcineurin.
      H. Caohuy and H. B. Pollard, unpublished data.
      Then, with the reduction of free Ca2+ concentration, the GDP-bound, unphosphorylated ANX7 releases Ca2+ and returns to the inactive form, and the cycle can recur.
      Figure thumbnail gr8
      Figure 8ANX7 membrane fusion cycle regulated by Ca2+, GTP , and PKC. This model schematically illustrates that ANX7 oscillates between two major transitional states. In the “off” state and low Ca2+ concentrations, ANX7 is inactive and exists in the favored GDP-bound form. Upon elevation of Ca2+, GDP bound to ANX7 can be replaced with GTP and this GTP-bound form is optimally phosphorylated by PKC, activating ANX7 (the “on” state), which drives membrane fusion much more efficiently. Then, with the reduction in the free Ca2+concentration, the off state complex is reformed, and the cycle can recur. See “Discussion” for a detailed explanation.
      In summary, the present observations on ANX7 are remarkably congruent with the original exocytotic model of Gomperts (
      • Lillie T.H.W.
      • Gomperts B.D.
      ). The ANX7 data are consistent with the concept that the stimulatory actions of Ca2+, GTP, and PKC converge on ANX7 to drive membrane fusion activity occurring during exocytosis. To further support such an inference, we have recently found that botulinum neurotoxin type C, which is a zinc-dependent protease and a specific inhibitor of exocytosis (
      • Dolly J.O.
      • de Paiva A.
      • Foran P.
      • Lawrence G.
      • Daniels-Holgate P.U.
      • Ashton A.C.
      ), efficiently cleaves ANX7 both in vitroand in permeabilized chromaffin cells. This proteolytic activity is concurrent with botulinum neurotoxin type C-dependent inhibition of ANX7 membrane fusion activity in vitro, and with inhibition of catecholamine secretion in vivo, respectively (

      Caohuy, H., and Pollard, H. B. (2002) Ann. N. Y. Acad. Sci., in press

      ). These recent findings significantly parallel the proteolytic effect of this toxin on syntaxin (
      • Schiavo G.
      • Shone C.C.
      • Bennette M.K.
      • Scheller R.H.
      • Montecucco C.
      ) and SNAP-25 (
      • Blasi J.
      • Chapmann E.R.
      • Yamasaki S.
      • Binz T.
      • Niemann H.
      • Jahn R.
      ), which are protein components of the SNARE hypothesis (
      • Rothman J.E.
      • Orci L.
      ). Inasmuch as the identification of SNARE proteins as targets for botulinum neurotoxins has been taken as prima facie evidence favoring the SNARE hypothesis for exocytotic membrane fusion, the apparent role of ANX7 in the exocytotic membrane fusion process thus cannot be excluded.

      Acknowledgments

      We thank Dr. Cathy Jozwik for preliminary reading of the manuscript.

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