Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain.

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.

Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase.

We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or beta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 or 3 days later. Application of 8-Br-cGMP caused decreases in calcium channel currents in cells expressing the alpha(1C) subunit, whether or not a beta subunit was co-expressed. No inhibition of currents by 8-Br-cGMP was observed in the presence of the protein kinase G inhibitor KT5823. Substitutions of serine residues by alanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subunit. As for wild type, the mutant S1371A exhibited inhibition of calcium channel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type channels but not for the mutant S533A. These results indicate that cGMP causes inhibition of L-type calcium channel currents by phosphorylation of the alpha(1C) subunit at position Ser(533) via the action of protein kinase G.

Investigating the role played by protein-lipid and protein-protein interactions in the membrane association of the p120GAP CaLB domain.

The GTPase activating protein, p120GAP, contains an amino acid sequence motif called the Ca2+-dependent lipid binding domain (CaLB) which mediates a protein-protein interaction between p120GAP and annexin VI and also binds to negatively charged phospholipids. Because membrane association of p120GAP is important for the regulation of p21 Ras activity, we have studied the roles played by Ca2+, phospholipids and annexin VI in the membrane association of p120GAP. Here we demonstrate that a truncated CaLB domain GST fusion protein (GSTGAP618-632), lacking the ability to bind to phospholipids, is able to bind to rat fibroblast membranes in a Ca2+- and concentration-dependent manner. In addition, this fusion protein also binds to annexin VI in an amino acid sequence specific but Ca2+ independent manner. Also, when bound to annexin VI in the presence of Ca2+, this fusion protein has the ability to co-bind to phosphatidylserine vesicles. Thus, annexin VI may simultaneously mediate an interaction with p120GAP and also an interaction with membrane phospholipids. This may in part explain the mechanism by which p120GAP associates with membranes in response to Ca2+ elevation and suggests the potential importance of annexin VI in the regulation of p21 Ras and the role CaLB domains may play in the specific recognition of cellular membranes.

Points of convergence between Ca2+ and Ras signalling pathways.

p21 Ras proteins play a critical role in the regulation of cellular growth and differentiation. In addition, Ras and proteins which regulate Ras activity have been implicated in long-term memory consolidation and long-term potentiation processes. Over the last few years, much evidence has emerged which indicates that changes in cytoplasmic Ca2+ levels can regulate Ras protein activity and subsequent biological function. Also, Ras proteins themselves can modulate intracellular Ca2+ levels by regulating both Ca2+ release and Ca2+ influx processes. Here we examine the signalling components which regulate Ras activity and, in particular, consider points of convergence between intracellular Ca2+ and p21 Ras signalling processes. In addition, we consider the possible biological consequences resulting from the integration of these signalling pathways and highlight the importance of our understanding protein protein interactions. Finally, we discuss the possibility of protein-protein interactions mediated via Ca2+-responsive structural domains, such as the C2 and IQ domains, playing important roles in Ca2+-dependent Ras functions yet to be established.

The Ca2+-dependent lipid binding domain of P120GAP mediates protein-protein interactions with Ca2+-dependent membrane-binding proteins. Evidence for a direct interaction between annexin VI and P120GAP.

The CaLB domain is a 43-amino acid sequence motif found in a number of functionally diverse signaling proteins including three Ras-specific GTPase activating proteins (GAPs). In the Ras GTPase activating protein, P120(GAP), this domain has the ability to confer membrane association in response to intracellular Ca2+ elevation. Here we have isolated three proteins, p55, p70, and p120, which interact with the P120(GAP) CaLB domain in vitro. We identify p70 as the Ca2+-dependent phospholipid-binding protein annexin VI. Using co-immunoprecipitation studies, we have shown that the interaction between P120(GAP) and annexin VI is also detectable in rat fibroblasts, suggesting that this interaction may have a physiological role in vivo. Thus, the CaLB domain in P120(GAP) appears to have the ability to direct specific protein-protein interactions with Ca2+-dependent membrane-associated proteins. In addition, annexin VI is known to have tumor suppressor activity. Therefore, it is possible that the interaction of annexin VI with P120(GAP) may be important in the subsequent modulation of p21(ras) activity.

Mutation-deletion analysis of a Ca(2+)-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras.

p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.

CaLB: a 43 amino acid calcium-dependent membrane/phospholipid binding domain in p120 Ras GTPase-activating protein.

CaLB was originally observed as a conserved sequence motif in various calcium-responsive signalling proteins and also in p120 Ras GTPase activating protein (p120GAP) (Clark et al. Cell 65: 1043-1051, 1991). Here we show the 43 residue CaLB motif in p120GAP is a functional protein domain that when expressed as a fusion protein in vitro confers Ca(2+)-dependent interactions with cellular membranes and phosphatidylserine and phosphatidylinositol vesicles. p120GAP, but not a mutant lacking the CaLB domain, associates with the particulate fraction of cells in response to elevated intracellular Ca2+ suggesting that p120GAP may be regulated in part by calcium signals. Addition of the p120GAP CaLB domain was able to restore transforming activity and particulate localization to an otherwise transformation-defective and cytosolic mutant v-Sre tyrosine kinase. The CaLB domain appears to be a prevalent protein module that may affect the molecular interactions and subcellular localization of signalling proteins.

Interactions between inositol tris- and tetrakis-phosphates. Effects on intracellular Ca2+ mobilization in SH-SY5Y cells.

The potential Ca2(+)-releasing activity of the inositol tetrakisphosphates Ins(1,3,4,6)P4 and DL-Ins(1,4,5,6)P4 and the inositol pentakisphosphate Ins(1,3,4,5,6)P5 and their effect on Ins(1,4,5)P3- and DL-Ins (1,3,4,5)P4-mediated Ca2+ release were examined in permeabilized SH-SY5Y human neuroblastoma cells. Neither DL-Ins(1,4,5,6)P4 nor Ins(1,3,4,5,6)P5 exhibit Ca2(+)-releasing activity at concentrations up to 10 microM, but Ins(1,3,4,6)P4 releases Ca2+ dose-dependently, with an EC50 value (conen, giving half-maximal effect) of 5.92 +/- 0.47 microM. Maximal response by this tetrakisphosphate (49 +/- 2.5%) is significantly less than that seen with Ins(1,4,5)P3 (60 +/- 3%) and is achieved at a concentration of 30 microM. In the presence of this concentration of Ins(1,3,4,6)P4 the EC50 value for Ins(1,4,5)P3-mediated Ca2+ release increases from 0.12 +/- 0.02 microM to 2.11 +/- 0.51 microM, providing evidence that this naturally occurring inositol tetrakisphosphate may recognize and exhibit its Ca2(+)-releasing activity via the Ins(1,4,5)P3 receptor. DL-Ins(1,3,4,5)P4, however, at its maximally effective concentration (10 microM) does not significantly affect Ins(1,4,5)P3-mediated Ca2+ release, and therefore appears to mediate its Ca2(+)-mobilizing action through a receptor distinct from that for Ins(1,4,5)P3.

Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells.

Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.

Metformin treatment of lean and obese Zucker rats modulates the ability of glucagon and insulin to regulate hepatocyte adenylate cyclase activity.

Glucagon stimulated adenylate cyclase activity some 21-fold in liver membranes from lean (Fa/Fa) and some 20-fold in membranes from obese (fa/fa) Zucker rats, with constants yielding half-maximal activation (Ka values) of 12.6 and 120.1 nmol/l respectively. Treatment of animals with the biguanide drug metformin (N',N'-dimethylbiguanide) decreased the ability of glucagon to stimulate this enzyme to some 16-fold for both the lean and obese animals and reduced the Ka values for activation of this enzyme by glucagon to 6.3 and 60.9 nmol/l respectively. Insulin inhibited glucagon-stimulated adenylate cyclase activity by some 24% in liver membranes from lean animals and some 17% in liver membranes from obese animals, with constants yielding half-maximal inhibition (Ki values) of 110 and 160 nmol/l respectively. The ability of insulin to inhibit the adenylate cyclase activity, from obese but not lean animals, was attenuated when insulin concentrations over 5 nmol/l were employed. Treatment of animals with metformin profoundly altered the sensitivity of adenylate cyclase to inhibition by insulin, with inhibition being increased to some 32% using liver membranes from either lean or obese animals. Values of Ki for this inhibitory action of insulin were 520 and 500 nmol/l using membranes from the lean and obese animals respectively, and no reduction in the ability of insulin, at concentrations over 5 nmol/l, to inhibit adenylate cyclase activity was observed using membranes from obese animals. Metformin also changed the kinetics of inhibition of adenylate cyclase by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon.

Brief exposure of hepatocytes to glucagon, angiotensin or the protein kinase C activator TPA (12-O-tetradecanoylphorbol 13-acetate) caused the inactivation of the inhibitory guanine nucleotide regulatory protein Gi. Glucagon-mediated desensitization of glucagon-stimulated adenylate cyclase activity was seen in hepatocytes from both normal rats and those made diabetic with streptozotocin, where Gi is not functionally expressed. Normal glucagon desensitization was seen in hepatocytes from young animals, 6 weeks of age, which had amounts of Gi in their hepatocyte membranes which were some 45% of that seen in mature animals (3.4 pmol/mg of plasma-membrane protein). Streptozotocin-induced diabetes in young animals abolished the appearance of functional Gi in hepatocyte plasma membranes. Pertussis-toxin treatment of hepatocytes from both normal mature animals and those made diabetic, with streptozotocin, blocked the ability of glucagon or angiotensin or TPA to elicit desensitization of adenylate cyclase. The isolated B (binding)-subunit of pertussis toxin was ineffective in blocking desensitization. Neither induction of diabetes nor treatment of hepatocytes with pertussis toxin inhibited the ability of glucagon and angiotensin to stimulate the production of inositol phosphates in intact hepatocytes. Thus (i) Gi does not appear to play a role in the molecular mechanism of glucagon desensitization in hepatocytes, (ii) the G-protein concerned with receptor-stimulated inositol phospholipid metabolism in hepatocytes appears not to be a substrate for the action of pertussis toxin, (iii) in intact hepatocytes, treatment with glucagon and/or angiotensin can elicit the inactivation of the inhibitory G-protein Gi, and (iv) pertussis toxin blocks desensitization by a process which does not involve Gi.

Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) Zucker rats: loss of functional Gi and abnormal Gs function.

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.

The phorbol ester TPA prevents the expression of both glucagon desensitisation and the glucagon-mediated block of insulin stimulation of the peripheral plasma membrane cyclic AMP phosphodiesterase in rat hepatocytes.

The phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) causes a dose-dependent inhibition of the glucagon-stimulated adenylate cyclase activity expressed in plasma membranes isolated from TPA-treated hepatocytes. However, no observable inhibitory effect of TPA on adenylate cyclase activity was observed in cells which had been exposed to glucagon for 5 min, prior to isolation, to desensitise adenylate cyclase. The degree of inhibition of adenylate cyclase elicited by both glucagon desensitisation and TPA treatment of hepatocytes was identical. Pre-treatment of hepatocytes with TPA was also found to prevent glucagon from blocking insulin's activation of the peripheral plasma membrane cyclic AMP phosphodiesterase in intact hepatocytes. TPA treatment also inhibited the ability of cholera toxin to activate the peripheral cyclic AMP phosphodiesterase in intact hepatocytes. It is suggested that in these particular instances TPA and glucagon elicit mutually exclusive processes rather than TPA mimicking glucagon desensitisation per se.