Joachim Krebs

Max Planck Institute for Biophysical Chemistry | MPIBPC · Department of NMR-based Structural Biology

In this review, I summarize the present knowledge of the structural and functional properties of the mammalian plasma membrane calcium pump (PMCA). It is outlined how the cellular expression of the different spliced isoforms of the four genes are regulated under normal and pathological conditions.

Ca²⁺ is a carrier of signals that are essential to cell life. The control of its cellular homeostasis demands an elaborate system of channels, exchangers, pumps and Ca²⁺-binding proteins that maintain free Ca²⁺in the cytosol in the 100–300 nM range. Many Ca²⁺ binding proteins process the Ca²⁺ signal and transmit it to target enzymes/proteins. Ca²⁺...

Dedicated to Professor Duilio Arigoni on the occasion of his 75th birthday Calcium may have a static, structure-stabilizing role in biological organs like the bones and the teeth, or may fulfill a dynamic function in cells as a regulator of signal-transduction pathways. This is made possible by the properties of the Ca 2 ion (e.g., high dehydration...

Thyroid hormones influence brain development through regulation of gene expression. Ca ²⁺ -dependent gene expression is a major pathway controlled by the Ca ²⁺ /calmodulin-dependent protein kinase IV (CaMKIV) which in turn is induced by the thyroid hormone T3 as also demonstrated in a mouse embryonic stem cell line. In addition, T3 is controlling t...

This book describes the newest discoveries on calcium signaling occurring at the cellular and intracellular membranes, often exerted in so called microdomains. Calcium entry and release, its interaction with proteins and resulting events on proteins and organelles are comprehensively depicted by leading experts in the field. Knowledge about details...

In this chapter the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to the structural and functional properties of PMCA, the tissue distribution of the different isoforms, including their differences during development. The importance of PMCA for regulating Ca2+signal...

Ca2+ is a carrier of signals that are essential to cell life. The control of its cellular homeostasis demands an elaborate system of channels, exchangers, pumps, and Ca2+-binding proteins that maintain free Ca2+in the cytosol in the 100–300 nM range. Many Ca2+-binding proteins process the Ca2+ signal and transmit it to target enzymes/proteins. Ca2+...

Thyroid hormones influence brain development through regulation of gene expression. This is especially true for Ca2 +-dependent regulation since a major pathway is controlled by the Ca2 +/calmodulin-dependent protein kinase IV (CaMKIV) which in turn is induced by the thyroid hormone T3. In addition, CaMKIV is involved in regulation of alternative s...

Calcium carries messages to virtually all important functions of cells. Although already active in unicellular organisms its role has become universally important after the transition to multicellular life. In this mini review we explore how calcium ended up in this privileged position. Most likely Its unique coordination chemistry has been decisiv...

Cellular Ca(2+) homeostasis is maintained through the integrated and coordinated function of Ca(2+) transport molecules, Ca(2+) buffers and sensors. These molecules are associated with the plasma membrane and different cellular compartments, such as the cytoplasm, nucleus, mitochondria, and cellular reticular network, including the endoplasmic reti...

Calcium is a very versatile second messenger involved in the regulation of a variety of different cellular processes. This is made possible due to the binding of Ca2+ to a great variety of different calcium-binding proteins, such as calcium-buffering or calcium-sensing proteins; the latter are also known as EF-hand containing proteins. In addition,...

Ca(2+)-signaling, alternative splicing, and stress responses by the endoplasmic reticulum are three important cellular activities which can be strongly interconnected to alter the expression of protein isoforms in a tissue dependent manner or during development depending on the environmental conditions. This integrated network of signaling pathways...

ALG-2 is a highly conserved calcium binding protein in the cytoplasm which belongs to the family of penta-EF hand proteins. Recently, we showed that ALG-2 is interacting with RBM22, a highly conserved spliceosomal nuclear protein (Montaville et al. Biochim. Biophys. Acta 1763, 1335, 2006; Krebs, Biochim. Biophys. Acta 1793, 979, 2009). In NIH 3T3 c...

In this review the influence of calcium signaling on the regulation of alternative splicing is discussed with respect to its influence on cell- and developmental-specific expression of different isoforms of the plasma membrane calcium pump (PMCA). In a second part the possibility is discussed that due to the interaction of the calcium-binding prote...

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state...

The Na+/Ca2+ exchanger (NCX) is a transmembrane protein of the plasma membrane involved in calcium homeostasis. It is a low-affinity, high-capacity electrogenic antiporter that extrudes Ca2+ against its concentration gradient at the expense of the inward-directed sodium gradient. Two different families of NCX exist, one independent of K+ (NCX), the...

Calcium is of pivotal importance for many biological processes. It may have a rather static, structure-stabilizing role, or it may participate as one of the second messengers of the cell in signal transduction pathways, fulfilling a more dynamic function. This is made possible by some specific properties of the Ca2+ ion (e.g. high dehydration rate,...

The Na+/Ca2+ exchanger (NCX) is a transmembrane protein of the plasma membrane involved in calcium homeostasis. It is a low-affinity, high-capacity electrogenic antiporter that extrudes Ca2+ against its concentration gradient at the expense of the inward-directed sodium gradient. Two different families of NCX exist, one independent of K+ (NCX), the...

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22....

Calcium is of pivotal importance for many biological processes. It may have a rather static, structure-stabilizing role, or it may participate as one of the second messengers of the cell in signal transduction pathways, fulfilling a more dynamic function. This is made possible by some specific properties of the Ca²⁺ ion (e.g. high dehydration rate,...

Calcium may have a static, structure-stabilizing role in biological organs like the bones and the teeth, or may fulfill a dynamic function in cells as a regulator of signal-transduction pathways. This is made possible by the properties of the Ca2+ ion (e.g., high dehydration rate, great flexibility in coordinating ligands, largely irregular geometr...

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids that modulates the activity of the reticular Ca(2+) ion pump. We recently solved the three-dimensional structure of chemically synthesized, unphosphorylated, monomeric PLN (C41F) by high-resolution nuclear magnetic resonance spectroscopy in chloroform/methanol. The structure is...

During the development of an organism, cell proliferation, differentiation and cell death are tightly balanced, and are controlled by a number of different regulators. Alterations in this balance are often observed in a variety of human diseases. The role of Ca(2+) as one of the key regulators of the cell is discussed with respect to a recently dis...

During the development of an organism cell proliferation, differentiation and cell death are tightly balanced, and are controlled by a number of different regulators. Alterations in this balance are often observed in a variety of human diseases. The role of Ca(2+) as one of the key regulators of the cell is discussed with respect to two recently di...

The structure of a novel oxido-aminoxyl (='nitronyl nitroxide') biradical, 2,2′-(1-oxidopyridine-2,6-diyl)bis [4,5-dihydro-4,4,5,5-tetramethyl-3-oxido-1H-imidazol-1-oxyl] hydrate (1·H 2O) was established by X-ray analysis in the solid state: monoclinic, space group P2 1c, Z = 4 with a = 12.621(4), b = 15.704(5), and c = 13.001(4) Å, and β = 115.202...

Phospholamban (PLN), an amphipathic intrinsic membrane protein of 52 amino acids, is the modulator of the Ca2+ pump of cardiac, slow-twitch, and smooth-muscle sarcoplasmic reticulum. In response to β-adrenergic stimulation, it becomes phosphorylated at Ser16 and/or Thr17, and dissociates from the pump, which, in turn, achieves its full activity. He...

Protein-dependent kinases are key regulators of many cellular events. One important class, the calmodulin-dependent protein kinases (CaMkinases), is dependent on changes of the intracellular concentrations of the second messenger Ca2+. In this chapter the structural and functional properties of CaMkinases are summarized. The different kinases known...

Volume III of this series deals with cellular calcium homeostasis aspects and structural properties of extra- and intracellular calcium-binding proteins are described. The structure/function relationship of calcium-transport proteins, and the role of calcium as a regulator of the activity specific protein kinases and phosphatase(s) are also describ...

The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total...

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In this chapter various aspects of apoptosis or programmed cell death (PCD) influenced by calcium as a mediator of signal transduction have been reviewed. Attention has been focused on recently described calcium-binding proteins such as ALG-2 or on a new calcium/calmodulin-dependent kinase, the death associated protein kinase or DAP-kinase. Both pl...

Calmodulin-dependent protein kinase IV (CaMKIV) is a key mediator of Ca2+-induced gene expression. This serine/threonine kinase is itself activated by a calmodulin kinase kinase. In the present contribution the gene structure, regulation of activity, the role in Ca2+-dependent gene expression, and the hormonal induction and controlled expression of...

In a primary cell culture system of fetal rat brain, the calmodulin-dependent protein-kinase IV (CaMKIV) could be induced by the thyroid hormone T3 in a time- and concentration-dependent manner, provided the tissue was excised not later than day 15 of gestation (E15) (Krebs et al., J. Biol. Chem. 271, 11055, 1996). We report here that in the fetal...

This chapter focuses on the Ca2+-pump of the plasma membrane and the molecular characterization of the structural and functional aspects of this enzyme. The chapter also presents the mechanistic and regulatory properties and discusses the structure and distribution of the different isoforms resulting from different genes or because of alternative s...

The expression of calmodulin kinase IV (CaMKIV) can be induced by the thyroid hormone T3 in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system which can grow and differentiate under chemically defined conditions (Krebs et al. (1996) J. Biol. Chem. 271...

This communication reports the specific induction of calmodulin kinase IV by the thyroid hormone 3,3′,5-triiodo-L-thyronine (T) in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system, which can grow and differentiate under chemically defined conditions...

The structure of the apo form of calcyclin, a member of the S100 family of calcium-binding proteins, reveals a novel dimer fold that may reflect the presence of a new interface for target protein recognition.

Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-A...

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca2+-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (pept...

The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains t...

The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains t...

The plasma membrane Ca2+-ATPase is one of the enzymes which are regulated by calmodulin (for a recent review see Carafoli et al., 1988). The interaction between the two proteins has been studied in de tail. The CaM-binding domain of the ATPase has recently been identified (James et al., 1988, Verma et al., 1988), and it has been shown that the Ca2+...

Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca{sup 2+} pump were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its...

The activity of the membrane-bound and the purified erythrocyte Ca²⁺-ATPase in the absence of calmodulin was stimulated by calpain digestion but could be further increased to maximal levels by calmodulin (CaM). Thus, CaM sensitivity was retained by the digested ATPase, at least at short times of incubation. In membranes digested at higher temperatu...

The isolation of a novel complementary DNA (cDNA) clone coding for human calmodulin (CaM) is reported. Although it encodes a protein indistinguishable from the only known higher vertebrate calmodulin, its nucleotide sequence varies extensively from that of two previously reported human CaM cDNAs (Wawrzynczak and Perham, 1984; SenGupta et al., 1987)...

Calmodulin and related Ca2+ -binding proteins were characterized using different labeling, chromatographic and spectroscopic techniques. Ca2+-binding to members of the so-called “EF-hand”- or “helix-loop-helix”-model protein family induced conformational changes, thereby exposing hydrophobic sites in some of them. These sites were identified using...

Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degra...

This chapter elaborates a comparison made of the characteristics of the activations by acidic phospholipids and controlled trypsinolysis. The pattern of trypsin proteolysis is described in some detail, with particular attention to the reconstitution of fragments in liposomes and to the effects of trypsin on the calmodulin binding domain of the mole...

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodie...

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodie...

Different conformational states of the purified plasma membrane Ca2+-ATPase from pig erythrocytes have been detected by circular dichroism (CD) and fluorescence spectroscopy. The helical content of the enzyme decreased by about 10% in the transition from the Ca2+ high-affinity form (10 microM free Ca2+ = E1 state) to the VO4(3-)-inhibited state (20...

Various Ca2+-antagonists and related compounds were probed for possible anti-calmodulin properties. Some of them efficiently inhibit calmodulin dependent activity (the plasma membrane Ca2+-ATPase and the cyclic nucleotide phosphodiesterase). The I50-values for the most potent inhibitors varied between 15 and 30 uM. Using fluorescence spectroscopy a...

Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engine...

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine ([125I]TID) labels apo-bovine alpha-lactalbumin but much less his Ca2+-form. The labeling of the apo-form is strong at protein concentrations of 0.5 mg ml-1 and increases with increasing concentration. Furthermore, increasing concentrations of NaCl, decrease t...

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID)labels apo-bovine α-lactalbumin but much less his Ca2+-form. The labeling of the apo-form is strong at protein concentrations of 0.5 mg ml−1 and increases with increasing concentration. Furthermore, increasing concentrations of NaCl, decrease the labe...

Reversed-phase, hydrophobic interaction, and ion-exchange high-performance liquid chromatography techniques have been used to separate different Ca2+-binding proteins and their proteolytic fragments. An alkali-stable ion-exchange column permitted the baseline separation of calmodulin fragments which differed only by one to three charged amino acids...

The highly hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) labels calmodulin in a Ca2+-dependent way [(1984) Biochemistry 23, 400–403]. Similar results are obtained in the presence of different cations depending on whether their properties are closely related to those of Ca2+. Labeling by [125I]TID i...

Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-...

The ADP/ATP translocator was selectively labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide in beef heart mitochondria, as reported previously for submitochondrial particles (Müller, M., Krebs, J. J. R., Cherry, R. J., and Kawato, S. (1982) J. Biol. Chem. 257, 1117-1120). The EMA binding was completely i...

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a highly hydrophobic, carbene-generating photoreactive probe, labels calmodulin and some of its proteolytic fragments in the Ca2+-bound conformation only. It is assumed that [125I]TID labels hydrophobic sites exposed by the binding of Ca2+. The finding offers a new and powerful means...

The purified Ca2+-pumping ATPase of the erythrocyte plasma membrane has been subjected to a controlled proteolytic treatment with trypsin. The treatment has been previously shown to shift the enzyme from low to high Ca2+ affinity in the absence of calmodulin. The treatment leads to the fragmentation of the ATPase molecule into a number of products...

The structural stability of calmodulin was studied by 1H-nuclear magnetic resonance spectroscopy under different denaturing conditions. The presence of Ca2+ stabilizes the structural properties of the native protein. In the absence of calcium the structural integrity of calmodulin can easily be affected by elevated temperatures or by high concentra...

Ca2+-induced conformational changes of calmodulin under a variety of different experimental conditions have been studied by 1H-nuclear magnetic resonance techniques. The assignment for Tyr-99 has been corrected. Ca2+ titration performed at pH 7.5 and greater than 9.5 apparently induces a different sequence of the protein folding process as can be m...

Submitochondrial particles were labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide. On sodium dodecyl sulfate-polyacrylamide gels, eosin fluorescence occurred in a single band of Mr approximately 30,000. The labeled band was identified as the ADP/ATP translocator, since EMA binding was completely inhibit...

The Ca 2+-ATPase of erythrocytes is the first Ca 2+-pumping enzyme ever identified in a plasma membrane. In the 13 years that have elapsed since its discovery, it has become obvious that Ca 2+-pumping ATPase are in all likelihood a constant feature of the plasma membranes, and constitute a family of enzymes in which subtle differences are superimpo...