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Calcium-binding proteins: beyond hands and loops
MISCELLANEA
The Second European Symposium on Calcium-Binding Proteins in Normal and Transformed Cells* followed the theme set by the first meeting in Brussels three years ago. Although predominantly a European meeting, the event brought together scientists from all corners of the world of calcium-binding proteins. It was satisfying to see that this vast area of scientific endeavour is flourishing, and that real advances have been made in many subjects. The majority of calcium-binding proteins fall into two easily recognizable classes: those with EF hands, and the annexins. The former, which have now been studied for three decades, exhibit an extraordinary diversity of function, exemplified by the multifunctional protein calmodulin. By contrast, the annexins are relative newcomers, and the subject of their functions continues to enjoy much controversy. The EF hand family of calciumbinding proteins (Ca2÷-BPs) now includes at least 29 subfamilies. These proteins contain between two and eight EF hands, each binding one calcium ion, and some contain EF hands spliced to other protein motifs. The studies by Robert Kretsinger (University of Virginia, USA) of this family contradict the assertion that introns have greatly facilitated domain shuffling and duplication in evolution: most of the introns are within domains, not between them, and their positions vary, implying that they were inserted after the definition of the subfamilies.
Calmodulin and calmodulinbinding proteins Many of the effects of Ca 2÷ in the cell are mediated through the ubiquitous and essential protein cal° modulin (CAM), which contains four EF hands. Interestingly, three separate CaM genes with divergent nucleotide sequences code for exactly the same amino acid sequence (presumably the divergence of nucleotide sequence minimizes the possibility of nonhomologous recombination). Emanuel Strehler (ETH Zurich, Switzerland) presented studies of the CaM III gene promoter. Sequence data revealed an abundance of promoter elements, and transfection experiments with a CaM III promoterreporter gene showed it to be a very strong 'housekeeping' promoter, as expected for a ubiquitous and essential gene. In fact, its expression may in some instances be regulated by negative factors. The most likely sites
Calcium-binding proteins: beyond hands and loops A. Louise Upton and Stephen E. Moss
of action for these 'silencing factors' are in the first intron, which is highly conserved in size and position between species. Strehler also reported an intronless human CaM-like gene. It encodes a CaM-like protein (CLP) of identical length to CaM but differing in 23 out of 148 amino acids. CaM and some CaM-binding proteins are located in the nuclei of some cell types. Oriel Bachs (University of Barcelona, Spain) reported the presence of calcineurin (CAN), a widely expressed CaM-dependent protein phosphatase, in the nuclei of rat liver cells. It specifically dephosphorylates two nuclear proteins: p37 and p45. The characterization of these proteins will be interesting since the role of Ca 2÷ in the nucleus is unclear. Tokichi Miyakawa and colleagues (Fermentation Technology, Higashi-Hiroshima, Japan) have identified and cloned two yeast homologues of mammalian CaN. Their functions are unknown and the only discernible effect on yeast cells of interrupting both genes was to make the cells sensitive to vanadate, an inhibitor of membrane ATPases. This suggests a role for CaN in regulating intracellular ion concentrations. Interestingly, and perhaps disappointingly, their disruption did not affect cell growth, suggesting that a third protein may exist with an overlapping function. CaM is a regulatory subunit of myosin light chain kinase. Paul Matrisan (Vanderbilt University, USA) presented his work on the unusual myosin light chain kinase gene. It contains a novel 'gene within a gene', i.e. the last three exons also encode a separate kinase-related protein. The two genes share the same open reading frame and the kinase-related protein has its own TATA box within the preceding intron of the myosin light chain kinase gene. The central role of Ca 2÷ and CaM in the cell cycle was emphasized by the work of Michael Whitaker (University College London, UK) on sea urchin eggs. Intracellular Ca2÷ 'transients' are the only signals necessary to cause both entry into and exit from mitosis. Ca 2÷ chelators injected
TRENDS IN CELL BIOLOGY VOL. 2 JUNE 1992
into these eggs can prevent mitosis, or arrest the egg in mitosis. Whitaker also reported studies on several cell cycle control proteins - cyclin, cdc25 and p34 cdc2 - in the context of their possible regulation by Ca2÷. It is well known that activation of the cyclinp34 ~dc2 complex is a prerequisite for entry into mitosis and that this is achieved through dephosphoryiation of a phosphotyrosine residue in the ATP-binding site of p34cdc2. It is interesting to note that cdc25, the phosphotyrosine phosphatase that activates cyclin-p34cdc2, is a substrate for CaM-dependent protein kinase, and inhibitors of this kinase can delay entry into mitosis. Phosphorylation of cdc25 by CaM-dependent protein kinase might thus explain the role of the premitotic Ca2÷ signal.
*Organized by R. Donarto, J. Haiech, C. Heizmannand R. Pochet. Marseille, France, 1-6 March 1992.
Annexins Steve Moss (University College London, UK) gave an overview of the annexin (Anx) family of proteins that bind Ca2÷ and phospholipids. There are now 12 members of this family, which have been cloned from species as diverse as human and Dictyostelium, and at least two of these exist as multigene families. In a field long dominated by Anx I and Anx II, the focus has now switched to Anx V since it is the only annexin for which the full crystal structure is known. Anita Lewit-Bentley (CNRS, Orsay, France) presented details of her work on Anx V. She reported that the protein is conformationally different when crystallized with Ca 2÷ compared with phosphatidylserine. Two Ca 2÷ ions are bound asymmetrically and cause the molecule to open on a hinge; binding a third causes the structure to close slightly. The crystal structure indicates that the four mainly c~-helical domains lie in the same plane, with a channel through the centre of the molecule surrounded by charged amino acid residues. In the model, the asymmetric charge distribution is proposed to disturb the phospholipid bilayer in such a way as to allow ions through. The three Ca2÷-binding loops extend from the convex side of the molecule and probably mediate close apposition of the protein
The authors are at the Department of Physiology, UniversityCollege London, Gower Street, London, UK WCIE 6BT.
© 1992 ElsevierSciencePublishersLtd (UK) 0962-8924/92/$05.00
175
MISCELLANEA
with the membrane. The theory that Anx V is a membrane channel for Ca 2÷ was much debated. This theory is controversial because the protein has no transmembrane regions and has not been shown to have Ca2÷channel activity in vivo. Alistair Aitken (NIMR, London, UK) reported the existence of two Anx V isoforms in bovine brain. They differ by only one amino acid (a substitution of Lys125 to Glu125), but this shifts their mobility on SDS-PAGE by 4 kDa, indicating a significant conformational change. The debate over the possible function of Anx V is in its infancy in comparison to the debate over the proposed anti-inflammatory role for Anx I. It is well known that phospholipase A 2 is central to the inflammatory response. It is inhibited in vitro by Anx I and indeed all annexins, although many believe that this may simply be due to substrate sequestration. Fran~oise Russo-Marie (INSERM, Paris, France) reported that the glucocorticoid analogue dexamethasone specifically induced Anx I secretion from lymphocytes and monocytes.
$100 proteins An overview of the $100 family of proteins was given by Rosario Donato (University of Perugia, Italy). There are three isoforms, with subunit structure c((z, c~13and 1313. Each chain has an EF hand at the C-terminus and an unconventional Ca 2+binding site at the N-terminus. The disulphide-linked 1313 dimer has neurotrophic activity in vivo. Linda
1 76
van Eldik (Vanderbilt University, USA) reported that the dimer causes an increase in intracellular Ca2+ concentration ([Ca2+]i) and hydrolysis of phosphoinositides in glial and neuronal cells. Intriguingly, the 1313-dimer also induced expression of early response genes such as fos and myc.
Ca2+-BPs in disease The Ca2÷-dependent dissociation of microtubules was discussed by E. Mandelkow (Max-Plank Research Unit, Hamburg, Germany). One of the microtubule-associated proteins (MAPs), tau, is phosphorylated by a CaM-dependent kinase. This phosphorylation produces a conformational change that causes it to run at a different position on SDSPAGE. Interestingly, this gel-shift form is found in tau proteins from patients with Alzheimer's disease. Jean-Pierre Brion (Free University of Brussels, Belgium) also presented work on tau in Alzheimer's disease. He hypothesized that a change in [Ca2+]i induced by a toxin activates a cascade of kinases that results in the abnormal phosphorylation of tau and, consequently, the loss of microtubules seen in brain neurons from patients with Alzheimer's disease. Thioredoxin reductase (TR) purified from metastatic amelanotic melanoma (TRAM) shows Michaelis-Menton kinetics in the reduction of 5,5'dithiobis2-nitrobenzoate (DTNB) as opposed to the sigmoidal kinetics displayed by TR from melanotic melanoma (TRM). A single calcium ion bound to a structure that is assumed to be an
EF hand in TR induces sigmoidal kinetics. High TR activity was found by Karin Schallreuter (University of Hamburg, Germany) to correlate very strongly with both metastatic progression of melanomas and with low [Ca2+]i. It was encouraging to hear that therapies based on TR analysis might improve survival rates. Some cancer patients have disorders caused by antibodies to tumour antigens that crossreact with epitopes in the nervous system. Arthur Polans (Good Samaritan Hospital and Medical Center, Oregon, USA) demonstrated that cancer-associated retinopathy is caused by autoantibody recognition of recoverin, the Ca2÷-dependent activator of photoreceptor guanylyl cyclase activity in retinal rods. Since neurological symptoms often precede cancer symptoms, the tumour epitope responsible could be used as a substrate for ELISAs in the diagnosis of cancer-associated retinopathy. Under normal physiological conditions, when a rod cell is stimulated, intracellular cGMP levels fall, closing the cGMP-gated Ca 2+ channel. The resulting decrease in [Ca2+]i causes the activation of guanylyl cyclase. This feedback mechanism is mediated by recoverin. Karl-Wilhelm Koch reported the phosphorylation of recoverin at Iow [Ca2+]i such as that found after illumination. He proposed that this modification potentiates the stimulation of guanylyl cyclase by recoverin at low [Ca2÷]i, allowing a rapid return to the resting state in the dark.
TRENDS IN CELL BIOLOGY VOL. 2 JUNE 1992
The Second European Symposium on Calcium-Binding Proteins in Normal and Transformed Cells* followed the theme set by the first meeting in Brussels three years ago. Although predominantly a European meeting, the event brought together scientists from all corners of the world of calcium-binding proteins. It was satisfying to see that this vast area of scientific endeavour is flourishing, and that real advances have been made in many subjects. The majority of calcium-binding proteins fall into two easily recognizable classes: those with EF hands, and the annexins. The former, which have now been studied for three decades, exhibit an extraordinary diversity of function, exemplified by the multifunctional protein calmodulin. By contrast, the annexins are relative newcomers, and the subject of their functions continues to enjoy much controversy. The EF hand family of calciumbinding proteins (Ca2÷-BPs) now includes at least 29 subfamilies. These proteins contain between two and eight EF hands, each binding one calcium ion, and some contain EF hands spliced to other protein motifs. The studies by Robert Kretsinger (University of Virginia, USA) of this family contradict the assertion that introns have greatly facilitated domain shuffling and duplication in evolution: most of the introns are within domains, not between them, and their positions vary, implying that they were inserted after the definition of the subfamilies.
Calmodulin and calmodulinbinding proteins Many of the effects of Ca 2÷ in the cell are mediated through the ubiquitous and essential protein cal° modulin (CAM), which contains four EF hands. Interestingly, three separate CaM genes with divergent nucleotide sequences code for exactly the same amino acid sequence (presumably the divergence of nucleotide sequence minimizes the possibility of nonhomologous recombination). Emanuel Strehler (ETH Zurich, Switzerland) presented studies of the CaM III gene promoter. Sequence data revealed an abundance of promoter elements, and transfection experiments with a CaM III promoterreporter gene showed it to be a very strong 'housekeeping' promoter, as expected for a ubiquitous and essential gene. In fact, its expression may in some instances be regulated by negative factors. The most likely sites
Calcium-binding proteins: beyond hands and loops A. Louise Upton and Stephen E. Moss
of action for these 'silencing factors' are in the first intron, which is highly conserved in size and position between species. Strehler also reported an intronless human CaM-like gene. It encodes a CaM-like protein (CLP) of identical length to CaM but differing in 23 out of 148 amino acids. CaM and some CaM-binding proteins are located in the nuclei of some cell types. Oriel Bachs (University of Barcelona, Spain) reported the presence of calcineurin (CAN), a widely expressed CaM-dependent protein phosphatase, in the nuclei of rat liver cells. It specifically dephosphorylates two nuclear proteins: p37 and p45. The characterization of these proteins will be interesting since the role of Ca 2÷ in the nucleus is unclear. Tokichi Miyakawa and colleagues (Fermentation Technology, Higashi-Hiroshima, Japan) have identified and cloned two yeast homologues of mammalian CaN. Their functions are unknown and the only discernible effect on yeast cells of interrupting both genes was to make the cells sensitive to vanadate, an inhibitor of membrane ATPases. This suggests a role for CaN in regulating intracellular ion concentrations. Interestingly, and perhaps disappointingly, their disruption did not affect cell growth, suggesting that a third protein may exist with an overlapping function. CaM is a regulatory subunit of myosin light chain kinase. Paul Matrisan (Vanderbilt University, USA) presented his work on the unusual myosin light chain kinase gene. It contains a novel 'gene within a gene', i.e. the last three exons also encode a separate kinase-related protein. The two genes share the same open reading frame and the kinase-related protein has its own TATA box within the preceding intron of the myosin light chain kinase gene. The central role of Ca 2÷ and CaM in the cell cycle was emphasized by the work of Michael Whitaker (University College London, UK) on sea urchin eggs. Intracellular Ca2÷ 'transients' are the only signals necessary to cause both entry into and exit from mitosis. Ca 2÷ chelators injected
TRENDS IN CELL BIOLOGY VOL. 2 JUNE 1992
into these eggs can prevent mitosis, or arrest the egg in mitosis. Whitaker also reported studies on several cell cycle control proteins - cyclin, cdc25 and p34 cdc2 - in the context of their possible regulation by Ca2÷. It is well known that activation of the cyclinp34 ~dc2 complex is a prerequisite for entry into mitosis and that this is achieved through dephosphoryiation of a phosphotyrosine residue in the ATP-binding site of p34cdc2. It is interesting to note that cdc25, the phosphotyrosine phosphatase that activates cyclin-p34cdc2, is a substrate for CaM-dependent protein kinase, and inhibitors of this kinase can delay entry into mitosis. Phosphorylation of cdc25 by CaM-dependent protein kinase might thus explain the role of the premitotic Ca2÷ signal.
*Organized by R. Donarto, J. Haiech, C. Heizmannand R. Pochet. Marseille, France, 1-6 March 1992.
Annexins Steve Moss (University College London, UK) gave an overview of the annexin (Anx) family of proteins that bind Ca2÷ and phospholipids. There are now 12 members of this family, which have been cloned from species as diverse as human and Dictyostelium, and at least two of these exist as multigene families. In a field long dominated by Anx I and Anx II, the focus has now switched to Anx V since it is the only annexin for which the full crystal structure is known. Anita Lewit-Bentley (CNRS, Orsay, France) presented details of her work on Anx V. She reported that the protein is conformationally different when crystallized with Ca 2÷ compared with phosphatidylserine. Two Ca 2÷ ions are bound asymmetrically and cause the molecule to open on a hinge; binding a third causes the structure to close slightly. The crystal structure indicates that the four mainly c~-helical domains lie in the same plane, with a channel through the centre of the molecule surrounded by charged amino acid residues. In the model, the asymmetric charge distribution is proposed to disturb the phospholipid bilayer in such a way as to allow ions through. The three Ca2÷-binding loops extend from the convex side of the molecule and probably mediate close apposition of the protein
The authors are at the Department of Physiology, UniversityCollege London, Gower Street, London, UK WCIE 6BT.
© 1992 ElsevierSciencePublishersLtd (UK) 0962-8924/92/$05.00
175
MISCELLANEA
with the membrane. The theory that Anx V is a membrane channel for Ca 2÷ was much debated. This theory is controversial because the protein has no transmembrane regions and has not been shown to have Ca2÷channel activity in vivo. Alistair Aitken (NIMR, London, UK) reported the existence of two Anx V isoforms in bovine brain. They differ by only one amino acid (a substitution of Lys125 to Glu125), but this shifts their mobility on SDS-PAGE by 4 kDa, indicating a significant conformational change. The debate over the possible function of Anx V is in its infancy in comparison to the debate over the proposed anti-inflammatory role for Anx I. It is well known that phospholipase A 2 is central to the inflammatory response. It is inhibited in vitro by Anx I and indeed all annexins, although many believe that this may simply be due to substrate sequestration. Fran~oise Russo-Marie (INSERM, Paris, France) reported that the glucocorticoid analogue dexamethasone specifically induced Anx I secretion from lymphocytes and monocytes.
$100 proteins An overview of the $100 family of proteins was given by Rosario Donato (University of Perugia, Italy). There are three isoforms, with subunit structure c((z, c~13and 1313. Each chain has an EF hand at the C-terminus and an unconventional Ca 2+binding site at the N-terminus. The disulphide-linked 1313 dimer has neurotrophic activity in vivo. Linda
1 76
van Eldik (Vanderbilt University, USA) reported that the dimer causes an increase in intracellular Ca2+ concentration ([Ca2+]i) and hydrolysis of phosphoinositides in glial and neuronal cells. Intriguingly, the 1313-dimer also induced expression of early response genes such as fos and myc.
Ca2+-BPs in disease The Ca2÷-dependent dissociation of microtubules was discussed by E. Mandelkow (Max-Plank Research Unit, Hamburg, Germany). One of the microtubule-associated proteins (MAPs), tau, is phosphorylated by a CaM-dependent kinase. This phosphorylation produces a conformational change that causes it to run at a different position on SDSPAGE. Interestingly, this gel-shift form is found in tau proteins from patients with Alzheimer's disease. Jean-Pierre Brion (Free University of Brussels, Belgium) also presented work on tau in Alzheimer's disease. He hypothesized that a change in [Ca2+]i induced by a toxin activates a cascade of kinases that results in the abnormal phosphorylation of tau and, consequently, the loss of microtubules seen in brain neurons from patients with Alzheimer's disease. Thioredoxin reductase (TR) purified from metastatic amelanotic melanoma (TRAM) shows Michaelis-Menton kinetics in the reduction of 5,5'dithiobis2-nitrobenzoate (DTNB) as opposed to the sigmoidal kinetics displayed by TR from melanotic melanoma (TRM). A single calcium ion bound to a structure that is assumed to be an
EF hand in TR induces sigmoidal kinetics. High TR activity was found by Karin Schallreuter (University of Hamburg, Germany) to correlate very strongly with both metastatic progression of melanomas and with low [Ca2+]i. It was encouraging to hear that therapies based on TR analysis might improve survival rates. Some cancer patients have disorders caused by antibodies to tumour antigens that crossreact with epitopes in the nervous system. Arthur Polans (Good Samaritan Hospital and Medical Center, Oregon, USA) demonstrated that cancer-associated retinopathy is caused by autoantibody recognition of recoverin, the Ca2÷-dependent activator of photoreceptor guanylyl cyclase activity in retinal rods. Since neurological symptoms often precede cancer symptoms, the tumour epitope responsible could be used as a substrate for ELISAs in the diagnosis of cancer-associated retinopathy. Under normal physiological conditions, when a rod cell is stimulated, intracellular cGMP levels fall, closing the cGMP-gated Ca 2+ channel. The resulting decrease in [Ca2+]i causes the activation of guanylyl cyclase. This feedback mechanism is mediated by recoverin. Karl-Wilhelm Koch reported the phosphorylation of recoverin at Iow [Ca2+]i such as that found after illumination. He proposed that this modification potentiates the stimulation of guanylyl cyclase by recoverin at low [Ca2÷]i, allowing a rapid return to the resting state in the dark.
TRENDS IN CELL BIOLOGY VOL. 2 JUNE 1992