Calcium effectors

Calmodulin and troponin C

Calmodulin

Calmodulin and its isoform troponin C are the most prominent Ca2 -sensing proteins in animal cells. Calmodulin itself is highly conserved and present

at significant levels in all eukaryotic cells. In mammalian brain it comprises about 1% of the total protein. Calmodulin, through its interaction with Ca2 , can cause the activation of more than 100 different enzymes, that are not necessarily themselves Ca2 -sensitive (see Table 8.1). The most important effectors of Ca2 -calmodulin are shown in Figure 8.1.

Calmodulin first came to light in association with cyclic nucleotide phosphodiesterase.3,4 It is an acidic protein of modest size (17 kDa), consisting of a single, predominantly helical polypeptide chain with four Ca2 -binding EF-hands, two at each end (Figure 8.2b). The affinities of the individual Ca2 binding sites are in the range 10 5–10 6 mol L 1 and adjacent sites bind Ca2 with positive cooperativity, so that attachment of the first Ca2 ion enhances the affinity of its neighbour. This has the effect of making the protein sensitive to small changes in the concentration of Ca2 within the signalling range. Ca2 - calmodulin itself has no intrinsic catalytic activity. Its actions depend on its close association with a target enzyme, in some instances, as in phosphorylase kinase and calcineurin, acting as a permanent component of a multisubunit complex.

In the absence of bound Ca2 , the helices of calmodulin pack so that their hydrophobic side chains are not exposed. In this form it is unable to interact with its targets (Figure 8.2a). Binding of Ca2 to the four sites induces a large conformational change causing the terminal helices to expose hydrophobic

Fig 8.1  Multiple signal transduction pathways activated by calmodulin. Calmodulin bound to Ca2 interacts with and activates many enzymes, opening up a wide range of cellular responses. Abbreviations: MAP-2, microtubule-associated protein-2; Tau, tubulin assembly unit.

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Fig 8.2  Three-dimensional structure of apocalmodulin, Ca2 -bound calmodulin, and calmodulin bound to a target peptide.

(a) Ca2 -free or apocalmodulin) (b) Calmodulin with four bound Ca2 ions (blue spheres). The terminal helices now expose hydrophobic residues (not indicated). (c) Calmodulin bound to a peptide corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase (1cfd,5 cll1,6 and 2bbm7).

surfaces and also a long central -helical segment (Figure 8.2b).8 Ca2 -bound calmodulin binds to its targets with high affinity (KD 10 9 mol L 1). To form the bound state, the central residues of the link region unwind from their -helical arrangement to form a hinge that allows the molecule to bend and wrap itself around the target. The N- and C-terminal regions approach each other and by their hydrophobic surfaces bind to it, rather like two hands holding a rope. This encourages the target sequence to adopt an -helical arrangement so that it occupies the centre of a hydrophobic tunnel (Figure 8.2c). The consequence of this interaction is a conformational change in the target, a state that persists only

as long as the Ca2 concentration remains high. When it falls, Ca2 dissociates and calmodulin is quickly released, inactivating the target. However, at least one important target protein is an exception to this rule. This is CaM-kinase II which can retain its active state after it has been activated by calmodulin (see below).

Troponin C

Troponin C, which regulates the interaction of actin and myosin in striated muscle, is effectively an isoform of calmodulin. It also possesses two pairs of Ca2 -binding EF-hands located at opposite ends of a peptide chain.

The affinities of these sites for Ca2 lie between 10 5 and 10 7 mol L 1. Its function is described below (page 237).

Kinases regulated by calmodulin

Among the enzymes controlled by calmodulin are a number of serine/ threonine kinases. These include phosphorylase kinase, myosin light chain kinase (MLCK), and members of the family of Ca2 -calmodulin-dependent kinases (CaM-kinases). Each of these interacts with calmodulin to convert a Ca2 signal into a phosphorylation signal.

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Fig 8.3  Calmodulin and Ca2 stimulate the breakdown of glycogen and inhibit its synthesis in skeletal muscle.

Phosphorylase kinase and glycogen synthase kinase

Phosphorylase kinase was the first protein kinase to be discovered.9 It is controlled by protein kinase A under -adrenergic stimulation and also through the effect of Ca2 on calmodulin (Figure 8.3). Even in the absence of Ca2 , calmodulin forms a permanent, integral subunit of phosphorylase kinase.10 When [Ca2 ] rises above the resting level, the kinase is activated both through its endogenous calmodulin and through the binding of an exogenous Ca2 -calmodulin. The enzyme catalyses glycogenolysis. To reinforce this, glycogen synthesis is simultaneously inhibited through the action of Ca2 -calmodulin

on glycogen synthase kinase, which phosphorylates and inactivates glycogen synthase (Figure 8.3). These themes will be developed further in Chapters 9 and 21 (page 679), where we shall see that phosphorylase kinase is also activated by a phosphorylation mediated by the second messenger cyclic AMP (cAMP).

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Myosin light chain kinase

MLCK functions in smooth muscle, where it phosphorylates the light chain of myosin II to activate contraction. This is described in more detail below

(see page 239).

Ca2 -calmodulin activated protein kinases

On the basis of in vitro phosphorylation assays the CaM kinases have been termed multifunctional, suggesting that each member of the family

phosphorylates a broad range of physiological targets. The range of substrates observed in vivo, however, is not so wide. All of the CaM kinases possess an autoinhibitory domain that folds over the active site. The calmodulin binding site overlaps this domain and when Ca2 -calmodulin binds, the domain is released and the inhibition is lifted. Like many other kinases, the CaM kinases are also regulated by phosphorylation.11 The different isoforms (CaMKI–IV) are as follows.

CaMKI

This is ubiquitously expressed in mammalian cells where it is predominantly cytoplasmic. Activation occurs through release of the autoinhibitory domain by Ca2 -calmodulin and also requires phosphorylation on a threonine in the activation loop by calmodulin kinase kinase. This upstream enzyme is itself activated by Ca2 -calmodulin. CaMKI has a number of targets that include the cystic fibrosis transmembrane regulator (CFTR) Cl channel. Activation loop phosphorylation may not be essential for some substrates.

CaMKII

a broad-spectrum serine/threonine kinase, CaMKII is the most prominent member of the family. It is widely expressed, with particularly high levels in brain. Unlike other kinases of this family, it has an additional association domain that lies C-terminal to its regulatory domain. This enables it to assemble as a multimeric cylindrical structure consisting of 8–12 copies of

the enzyme as subunits. CaMKII is encoded by four genes, , , , and ; the and subtypes are expressed only in brain. There are also a number of splice variants. As the multimeric structures can assemble as either homomers or heteromers, there is potential for considerable diversity.

Activation of CaMKII occurs when one of the subunits in a multimeric structure binds Ca2 -calmodulin. Its autoinhibitory domain is released from its active site and this allows it to phosphorylate the autoinhibitory domain of the neighbouring subunit at T286. (Note that unlike the other CaM kinases, this is not an activation loop phosphorylation.) Phosphorylation of the neighbouring subunit not only releases its autoinhibitory domain, it also increases its affinity for Ca2 -calmodulin by a factor of more than 1000. This effectively ‘traps’ a second Ca2 -calmodulin and the enzyme can then remain active or

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‘autonomous’ for tens of minutes after the Ca2 stimulus has been removed. It remains autonomous until the bound calmodulin is eventually released and the phosphate at T286 is cleaved by a phosphatase. Moreover, in the face of a train of spikes or oscillations, its level of activation, presumably related to the

number of subunits activated, is matched to the frequency and duration of the Ca2 spikes. This ability to decode Ca2 signals and to become autonomous is thought to underlie the role of CaMKII in learning and memory.

CaMKIII

The protein formerly known as CaMKIII specifically phosphorylates eukaryotic elongation factor 2 (eEF2), a GTPase necessary for the elongation step in protein translation. This kinase has no sequence similarity with the other members of the CaM kinase family and it is now termed eEF2 kinase.

CaMKIV

This is expressed in a limited range of tissues, principally neuronal cells, male germ cells, and lymphocytes, where it regulates gene expression by phosphorylating transcription factors. It is found mostly in the nucleus. It is activated on binding of Ca2 -calmodulin to the autoinhibitory domain, followed by autophosphorylation in the activation loop at by CaM kinase kinase. An important target is the transcription factor CREB which is activated by CaMKIV, but inhibited by CaMKII. CaMKIV can become partly Ca2 -calmodulin-independent when autophosphorylated.

Other Ca2 -calmodulin dependent enzymes

Plasma membrane Ca2 ATPase (PMCA)

The plasma membrane Ca2 ATPases that are responsible for Ca2 homeostasis in the cell were introduced in Chapter 7 (page 190). There are four isoforms. PMCA1 and 4 are ubiquitous, while PMCA2 and PMCA3 are predominant in neuronal cells.12 Together with the SERCAs and the Na -Ca2 exchangers, they maintain cytosol [Ca2 ] in the range 40–100 nmol L 1. Under these conditions, the Ca2 -affinity of the PMCAs is low (KD 10  mol L 1) and therefore they are barely active. When Ca2 is elevated, Ca2 -calmodulin binds to the C-terminal tail of the ATPase, increasing its affinity for Ca2 by a factor of 10, and as a consequence its activity. However, acidic phospholipids, such as phosphatidylserine and PI(4,5)P2, can also increase Ca2 -affinity to an even greater extent, implying that the most active pumps might be localized to membrane domains rich in these lipids. Other potential modulators also interact with PMCAs, such as calcineurin (see below).

Ras guanine nucleotide exchange factor

The principal signalling pathway through the GTPase Ras, described in Chapter 4, involves the targeting of the guanine nucleotide exchange factor Sos to the

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FKBP12 respectively). The drug–protein complex binds to calcineurin as shown in Figure 8.4 and prevents substrate from gaining access to the active site. In this way they prevent the activation of T lymphocytes through the dephosphorylation of the transcription factor NFAT. It should be noted that immunophilins are not restricted to cells of the immune system. They also influence cell growth and repair in nerves15 and they are abundant in other cells as components of ryanodine and IP3 receptor complexes (see page 200).

Another target of calcineurin is the inhibitory-1(I-1) subunit of protein phosphatase 1 (PP1). I-1 acts as an inhibitory pseudosubstrate and when it is dephosphorylated by calcineurin, it is released from the phosphatase and the inhibition is lifted. As PP1 has multiple targets, this expands the range of phosphoproteins that may be dephosphorylated when calcineurin is activated (see page 679).

The effective Ca2 affinity of calcineurin is high, allowing it to be activated by a mild increase in concentration. It then remains active until the level subsides. By contrast, CaMKII (see above) has low Ca2 affinity and responds most effectively to one or more sharp transients, after which it can remain autonomous for tens of minutes. Thus CaMKII responds best to the intense Ca2 transients that occur close to the mouths of open channels, but not to a mild more generalized elevation of Ca2 in the bulk cytosol. Conversely, calcineurin is most effectively activated by a sustained Ca2 increase in the cytosol, where its target transcription factors await dephosphorylation. Thus

the two effectors are sensitive to Ca2 signals that have different temporal and spatial characteristics. For example, in neurons undergoing mild stimulation, there will only be local activation of Ca2 -effectors, such as the CaM kinases, because Ca2 ions entering through channels are restricted to microdomains by local buffers and pumps. Alternatively, strong stimulation, generating a global and longer-lasting Ca2 increase, may activate transcription that leads to the modulation synaptic strength.

Ca2 -calmodulin-sensitive adenylyl cyclases and phosphodiesterase

Ca2 - and cAMP-mediated signalling pathways are linked through the Ca2 -dependent isoforms of adenylyl cyclase (AC) described in Chapter 5. AC1 and AC8 are activated by Ca2 -calmodulin, while AC5 and AC6 are inhibited by Ca2 , independently of calmodulin. The Ca2 -sensitive ACs occupy regions of the plasma membrane rich in cholesterol and glycosphingolipids (lipid rafts, see page 522). This keeps them close to the channels that admit Ca2 and to other signalling proteins. They also encounter molecules of cyclic nucleotide phosphodiesterase 1 (PDE1), a Ca2 - and calmodulin-dependent terminator of cAMP signalling. Consequently, when Ca2 enters neurons through voltage-dependent plasma membrane channels (e.g. L-type channels), it can stimulate AC1 or AC8 to produce cAMP, which is then broken

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