Activating ROCK1 somatic mutations in human cancer.

Cancer cells acquire characteristics of deregulated growth, survival and increased metastatic potential. Genetic mutations that provide a selective advantage by promoting these characteristics have been termed 'drivers,' whereas mutations that do not contribute to disease initiation/progression are termed 'passengers.' The advent of high-throughput methodologies has facilitated large-scale screening of cancer genomes and the subsequent identification of novel somatic mutations. Although this approach has generated valuable results, the data remain incomplete until the functional consequences of these mutations are determined to differentiate potential drivers from passengers. ROCK1 is an essential effector kinase downstream of Rho GTPases, an important pathway involved in cell migration. The Cancer Genome Project identified three nonsynonymous mutations in the ROCK1 gene. We now show that these somatic ROCK1 mutations lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy terminus combined with structural modeling provides an insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire 'driver' mutations that result in kinase activation.

Subtype identification and functional properties of inositol 1,4, 5-trisphosphate receptors in heart and aorta.

One of the major mechanisms by which hormones elevate intracellular Ca(2+)levels is by generating the second messenger inositol 1,4, 5-trisphosphate (InsP(3)), which activates a Ca(2+)channel (InsP(3)receptor) located in the endoplasmic reticulum (ER). This study undertakes to identify the InsP(3)receptor subtypes (isoforms) in heart and aorta and to characterize their functional properties. The InsP(3)receptor isoforms were identified from rat heart and aorta tissues using both reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA for the different isoforms and immunochemistry using InsP(3)receptor isoform-specific antibodies. Functional studies included ligand binding experiments using [(3)H]InsP(3)and InsP(3)-induced Ca(2+)release studies using Fluo-3 as the Ca(2+)sensing dye. All three isoforms of the InsP(3)receptor were identified using RT-PCR and immunochemical analyses. [(3)H]InsP(3)binding studies using microsomes derived from these tissues showed that heart had a 3-fold lower abundance of InsP(3)receptors than aorta, while both have considerably lower abundance than the well characterized cerebellar microsomes. The affinity of the InsP(3)binding to the receptor was also different in the three tissues. In cerebellum the K(d)was 60 nM, while aorta had a much higher K(d)of 220 nM. Heart microsomes, appeared to show two classes of binding affinity with K(d)s of 150 nM and 60 nM. Furthermore, the effects of free [Ca(2+)] on [(3)H]InsP(3)binding levels were also different for the three tissues. InsP(3)binding to both cerebellar and aorta microsomes decreased by 90% and 60%, respectively, above 30 nM free [Ca(2+)], while InsP(3)binding to heart was relatively insensitive to changes in [Ca(2+)]. At maximal InsP(3)concentrations, aorta microsomes were able to release about 5% of the accumulated Ca(2+), compared to 25% by cerebellar microsomes. Heart microsomes, however, showed only very little InsP(3)-induced Ca(2+)release ( <0.5%). The EC(50)concentration for InsP(3)-induced Ca(2+)release was 1.2 micro M for aorta while that for cerebellum was 0.3 micro M. Known agonists of the cerebellar InsP(3)receptor such as 3-deoxy InsP(3)and adenophostin A were also able to mobilize Ca(2+)from aorta microsomes. In addition, the competitive antagonist heparin and the non-competitive antagonists of the cerebellar InsP(3)receptor, tetracaine and tetrahexylammonium chloride, were also able to block InsP(3)-induced Ca(2+)release from aorta microsomes.

The mechanism of inhibition of the Ca2+-ATPase by mastoparan. Mastoparan abolishes cooperative ca2+ binding.

The amphiphilic peptide mastoparan, isolated from wasp venom, is a potent inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. At pH 7. 2, ATPase activity is inhibited with an inhibitory constant (Ki) of 1 +/- 0.13 microM. Mastoparan shifts the E2-E1 equilibrium toward E1 and may affect the regulatory ATP binding site. The peptide also decreases the affinity of the ATPase for Ca2+ and abolishes the cooperativity of Ca2+ binding. In the presence of mastoparan, the two Ca2+ ions bind independently of one another. Our results appear to support the model that describes the relationship between the two Ca2+ binding sites as "side-by-side," because this model allows the possibility of independent Ca2+ entry to the two sites. Mastoparan shifts the steady-state equilibrium between E1'Ca2 and E1'Ca2.P toward E1'Ca2.P, by possibly affecting the conformational change that follows ATP binding. The peptide also causes a reduction in the levels of phosphoenzyme formed from [32P]Pi. Some analogues of mastoparan were also tested and were found to cause inhibition of the Ca2+-ATPase in the range of 2-4 microM. The inhibitory action of mastoparan and its analogues appears dependent on their ability to form alpha-helices in membranes.

Biochemical mechanisms of calcium mobilisation induced by mastoparan and chimeric hormone-mastoparan constructs.

Ca2+ efflux, Ca(2+)-ATPase, and membrane permeability measurements were used to investigate the biochemical mechanisms of Ca2+ release induced by mastoparan (MP) and the chimeric hormone-MP constructs incorporating galanin (galparan) or vasopressin antagonist (M375 and M391) moieties. Comparative studies utilised preparations of porcine cerebellar microsomes and rabbit skeletal muscle sarcoplasmic reticulum (SR). MP and chimeric peptides galparan, M375 and M391 induce Ca2+ release over a range of concentrations from 0.3-10 microM. Comparison of MP and three chimeric, N-terminal extended, constructs indicates that N-terminal extension modifies the biological properties of MP, producing changes in efficacy which are enzyme-isoform-specific. Biochemical studies indicate that the chimeric analogues and MP inhibit Ca(2+)-ATPases and directly activate the ryanodine receptor (RyR) to release Ca2+ from both heavy SR (HSR) and microsomes. The same peptides have no effect on the InsP3 receptor (InsP3R). Other actions that include modest changes in membrane permeability may also contribute to the Ca(2+)-mobilising action of MP and chimeric constructs.

2-Hydroxycarbazole induces Ca2+ release from sarcoplasmic reticulum by activating the ryanodine receptor.

2-Hydroxycarbazole was shown to induce Ca2+ release from skeletal muscle and cardiac muscle sarcoplasmic reticulum at concentrations between 100-500 microM. This release was blocked by both 1 mM tetracaine and 30 microM ruthenium red which inhibit the ryanodine receptor or by pre-treatment with 10 mM caffeine which depletes the ryanodine receptor-containing Ca2+ stores. This, in addition to the fact that 2-hydroxycarbazole has little effect on Ca2+ ATPase activity, indicates that it activates Ca2+ release through the ryanodine receptor. The apparent EC50 value for release from both skeletal muscle and cardiac muscle sarcoplasmic reticulum was approximately 200 microM and maximal release occurred at 400-500 microM, making it approximately 20 times more potent than caffeine. The dose-dependency in the extent of Ca2+ release induced by 2-hydroxycarbazole was also apparently highly cooperative for both preparations. That 2-hydroxycarbazole was able to mobilize Ca2+ from non-muscle cell microsomes and in intact TM4 cells (which contain ryanodine receptors), makes this compound a more potent and commercially available alternative to caffeine in studying the role of this intracellular Ca2+ channel in a variety of systems.

The role of inorganic phosphate in regulating the kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release: a putative role for endoplasmic reticulum phosphate transporters.

The effects of phosphate and acylphosphonate phosphate transporter inhibitors were investigated on inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from cerebellar microsomes. Although neither changing the phosphate concentration nor adding phosphate transporter inhibitors affected the percentage (extent) of InsP3-induced Ca2+ release, they did, however, affect the transient kinetics of this process. InsP3-induced Ca2+ release is biphasic in nature, arising from two populations of InsP3-sensitive Ca2+ stores which either release Ca2+ in a fast or slow fashion. Altering phosphate concentration or adding phosphate transporter inhibitors appeared to affect only the fast phase component. We therefore suggest that these observations could be explained by the possibility that phosphate transporters only reside in the fast releasing InsP3-sensitive Ca2+ stores.

Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg.

1. The sea urchin egg homogenate is an ideal model to characterize Ca2+-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP3- and ryanodine-sensitive Ca2+-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. 2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+-releasing agent, NAADP, compared to inositol trisphosphate (InsP3) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. 3. NAADP-induced Ca2+-release was blocked by L-type Ca2+-channel blockers and by Bay K 8644, while InsP3- and cyclic ADPR-induced Ca2+-release were insensitive to these agents. L-type Ca2+-channel blockers did not displace [32P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion. 4. Similarly, a number of K+-channel antagonists blocked NAADP-induced Ca2+-release selectively over InsP3- and cyclic ADPR-induced Ca2+-release. Radioligand studies showed that these agents were not competitive antagonists. 5. As has been shown for InsP3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+-release mechanisms. 6. The presence of K+ was not essential for NAADP-induced Ca2+-release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP3-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca2+-release. While the alkaline condition permitted InsP3 and cyclic ADPR-induced Ca2+-release, the acidic condition completely hampered both Ca2+-release mechanisms. 7. The present results characterize pharmacologically and biochemically the novel Ca2+-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

Effects of thimerosal on the transient kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release from cerebellar microsomes.

Thimerosal, a thiol-reactive reagent, has been shown to increase the cytosolic Ca2+ concentration in a variety of cells by sensitizing inositol 1,4,5-trisphosphate (InsP3) receptors. Thimerosal can have both sensitizing (at concentrations of <2 microM) and inhibitory (at concentrations of >2 microM) effects on InsP3-induced Ca2+ release (IICR) from cerebellar microsomes. Transient kinetic studies were performed by employing a fluorimetric stopped-flow approach using fluo-3. IICR was found to be a bi-exponential process with a fast and a slow component. At a maximal InsP3 concentration (20 microM), the fast phase had a rate constant of 0.9 s-1 and the slow phase had a rate constant of 0.4 s-1. The amplitudes of the two phases were 60% and 40% respectively. When the rate constants for the two phases were plotted as Hill plots, the processes were found to be non-co-operative in both cases (Hill coefficient of 1.0), thus arguing for a simple mechanism linking InsP3 binding to channel opening. At a submaximal InsP3 concentration (0.2 microM), where the sensitizing effects of thimerosal are most pronounced, thimerosal increased the rate constants of both phases in a sigmoidal fashion, with a Hill coefficient of 4.0, suggesting that several cysteine residues (up to four) need to be modified in order for maximum sensitization to occur. The rate constants remained elevated even at thimerosal concentrations that inhibited IICR. The amplitude or extent of Ca2+ release was, however, elevated to a much greater extent in the slow phase, suggesting that the two phases respond differently. At maximal InsP3 concentrations, thimerosal has no effect upon the rate constants but inhibits the amplitude of Ca2+ release.

Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis.

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1+ and S1-), but only the S2- from of the S2 splice variant of the type I InsP3 receptor. PCR analysis was also used to identify InsP3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [3H]-InsP3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a Bmax of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The Kd for InsP3 binding to its receptor in testicular microsomes was 60 +/- 10 nM which was similar to that found for cerebellar microsomes (80 +/- 20 nM). InsP3-induced Ca2+ release (IICR) in testicular microsomes was found to have an EC50 (concentration which causes a half-maximal response) of 0.5 +/- 0.03 microM, also similar to that seen for cerebellar microsomes (0.3 microM). Maximal IICR occurred at about 20 microM InsP3, with up to 4% of total intracellular Ca2+ stores being mobilized as compared to between 10-30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s-1 at 30 microM InsP3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (approximately 1 s-1) and taken together with the binding data support the proposal that the receptor density/Ca2+ store is approximately 8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP3 analogues also confirmed similar behaviour for testicular InsP3Rs and cerebellar InsP3Rs.

Kinetic properties of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release.

Three endogenous molecules have now been shown to release Ca2+ in the sea urchin egg: inositol trisphosphate (InsP3), cyclic adenosine 5'-diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), a derivative of NADP. While the mechanism through which the first two molecules are able to release Ca2+ is established and well characterized with InsP3 and cADPR-activating InsP3 and ryanodine receptors, respectively, the newly described NAADP has been shown to release Ca2+ via an entirely different mechanism. The most striking feature of this novel Ca2+ release mechanism is its inactivation, since subthreshold concentrations of NAADP are able to fully and irreversibly desensitize the channel. In the present study we have investigated the fast kinetics of activation and inactivation of NAADP-induced Ca2+ release. NAADP was found to release Ca2+ in a biphasic manner, and such release was preceded by a pronounced latent period, which was inversely dependent on concentration. Moreover, the kinetic features of NAADP-induced Ca2+ release were not altered by pretreatment with low concentrations of NAADP, although the extent of Ca2+ release was greatly affected. Our data suggest that the inactivation of NAADP-induced Ca2+ release is an all-or-none phenomenon, and while some receptors have been fully inactivated, those that remain sensitive to NAADP do so without any change in kinetic features.

The effects of inositol 1,4,5-trisphosphate (InsP3) analogues on the transient kinetics of Ca2+ release from cerebellar microsomes. InsP3 analogues act as partial agonists.

An investigation of the effects of a number of inositol trisphosphate analogues on the transient kinetics of Ca2+ release from cerebellar microsomes was undertaken. All the analogues investigated could release the total Ca2+ content of the inositol 1, 4,5-trisphosphate (Ins(1,4,5)P3) mobilizable Ca2+ store; however, their potencies were substantially reduced compared to Ins(1,4,5)P3. The concentration required to induce half-maximal Ca2+ mobilization was 0.14 microM for Ins(1,4,5)P3, 1.8 microM for 3-deoxyinositol 1,4, 5-trisphosphate (3-deoxyInsP3), 1.0 microM for 2,3-dideoxyinositol 1, 4,5-trisphosphate (2,3-dideoxyInsP3), 24 microM for 2,3, 6-trideoxyinositol 1,4,5-trisphopshate (2,3,6-trideoxyInsP3), and 2.9 microM for inositol 2,4,5-trisphosphate (Ins(2,4,5)P3). In all cases and for all concentrations tested, the inositol trisphosphate analogues induced biphasic transient release of Ca2+, which could fit to a biexponential equation assuming two independent processes. The rate constants calculated for the release process were much larger for Ins(1,4,5)P3 than the other inositol trisphosphates (the fast phase rate constant varying from 0.3 to 1.6 s-1 and the slow phase from 0.01-0.5 s-1, at concentrations between 0.03 and 20 microM Ins(1,4,5)P3). The rate constants for all other inositol trisphosphates did not appear to exceed 0.4 s-1 for the fast phase and 0.1 s-1 for the slow phase at their highest concentrations tested. The maximum amplitudes for Ca2+ release by the two phases appeared to be similar for all inositol trisphosphates (approximately 45% for the fast phase and approximately 55% for the slow phase). On comparing the rate constants for Ca2+ release at inositol trisphosphate concentrations for the analogues which all induced the same extent of Ca2+ release, it was apparent that the rates of release were independent of the extent of Ca2+ release. As the extent of Ca2+ release can be related to degree of occupancy of the binding sites, it is evident that different analogues which occupy the binding site of the receptor to the same extent can induce Ca2+ to be released at different rates. We explain this conclusion in terms of partial agonism where inositol phosphates can induce two (or more) occupied states of the channel.

Conformational changes associated with activation of bee venom phospholipase A2.

Bee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A.J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A.J. and Moores, G.R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary structure with apparent increase in both alpha-helix and beta-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.

Inhibition of the cerebellar inositol 1,4,5-trisphosphate-sensitive Ca2+ channel by ethanol and other aliphatic alcohols.

The effects of ethanol and other aliphatic alcohols on the endoplasmic reticulum Ca2+ pump and the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channel were studied in pig cerebellar microsomes. Methanol, ethanol and propanol all stimulated ATP-dependent Ca2+ uptake, whereas butanol inhibited this process. Ethanol inhibited InsP3-induced Ca2+ release [half-maximal inhibition at 3.5%, v/v (600 mM)]. However, ethanol affected only the amount of InsP3-releasable Ca2+, without affecting the concentration of InsP3 required to induce half-maximal release. Other alcohols of longer chain length were more potent than ethanol at inhibiting InsP3-induced Ca2+ release, but none of the alcohols tested affected [3H]InsP3 binding to its receptor. Using stopped-flow techniques, measurements of the rate of InsP3-induced Ca2+ release in the preparation of pig cerebellar microsomes used in this study showed the kinetics to be monophasic, with a rate constant of 0.93s-1 at 20 microM InsP3. This rate constant was dependent upon InsP3 concentration, decreasing to 0.38s-1 at 0.25 microM InsP3. Ethanol was shown to reduce the fractional amount of InsP3-induced Ca2+ release without significantly affecting the rate constant for this process.

Alkali metal ion dependence of inositol 1,4,5-trisphosphate-induced calcium release from rat cerebellar microsomes.

The effects of the alkali metal ions Na+, K+, Rb+, and Cs+ on ATP-dependent Ca2+ uptake, [3H]Inositol 1,4,5-trisphosphate (InsP3) binding, and quantal InsP3-induced Ca2+ release were investigated using rat cerebellar microsomes. Both the ion species and concentration affected the ability of the microsomes to support Ca2+ uptake with K+ being mot effective (3.8 nmol of Ca2+/min/mg at 100 mM K+). The order of efficacy of the other ions was as follows: K+ > Na+ > Rb+ = Cs+ >> Li+. The binding of [3H]InsP3 to cerebellar microsomes was, however, affected little by the presence of these ions. All these alkali metal ions (except Li+) supported InsP3-induced Ca2+ release at concentrations above 25 mM; however, the extent of Ca2+ release (expressed as a percent Ca2+ release compared with that released by the ionophore A23187) was dependent upon the ion species present. Again K+ was more potent than the other ions at facilitating InsP3-induced Ca2+ release (order of efficacy: K+ > Rb+ > Na+ > Cs+), although the concentration of InsP3 required to induce half-maximal Ca2+ release (IC50) was not significantly altered. Over the ion concentration range tested (25-100 mM), the extent of InsP3-induced Ca2+ release with both K+ and Rb+ increased in a linear fashion, while Na+ showed only a slight increase and Cs+ showed no increase over this range. The effect of K+ concentration on quantal Ca2+ release was to alter the extent of release rather than the IC50 InsP3 concentration. Using stopped-flow techniques, the effects of InsP3 and K+ concentrations on the kinetics of InsP3-induced Ca2+ release were shown to exhibit a monoexponential process in this microsomal preparation. The rate constants for Ca2+ release increased with InsP3 concentration (0.11 s-1 at 0.02 microM InsP3 to 0.5 s-1 at 40 microM InsP3); however, the relationship between the fractional extent of release and rate constants for release did not change in a similar way with InsP3 concentration. Although the fractional extent of Ca2+ release increased with K+ concentration, the rate constants for release over this K+ concentration range were unaffected. This observation leads us to question the role of K+ as a counter ion required for Ca2+ release, and we therefore postulate a role for K+ (and the other alkali metal ions) as a "co-factor" required for channel opening.

Pharmacological modulators of the inositol 1,4,5-trisphosphate receptor.

Elevation of cytosolic calcium concentrations, induced by many neurotransmitters, plays a crucial role in neuronal function. Some neurotransmitters produce the second messenger InsP3 which activates an intracellular calcium channel (InsP3 receptor) usually located in the endoplasmic reticulum. This article undertakes a comprehensive survey of most pharmacological modulators of the InsP3 receptor so far reported. This review discusses in detail competitive antagonists, non-competitive antagonists and thiol reactive reagents, highlighting their modes of action and in some cases indicating drawbacks in their use.