Calmodulin has the Potential to Function as a Ca2+-Dependent Adaptor Protein

Calmodulin (CaM) is a versatile Ca²⁺-binding protein that regulates the activity of numerous effector proteins in response to Ca²⁺ signals. Several CaM-dependent regulatory mechanisms have been identified, including autoinhibitory domain displacement, sequestration of a ligand-binding site, active site reorganization, and target protein dimerization. We recently showed that the N- and C-lobes of animal and plant CaM isoforms could independently and sequentially bind to target peptides derived from the CaM-binding domain of Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1), to form a 2:1 peptide:CaM complex. This suggests that CaM might facilitate the dimerization of NtMKP1, although the dimerization mechanism is distinct from the previously described simultaneous binding of other target peptides to CaM. The independent and sequential binding of the NtMKP1 peptides to CaM also suggests an alternative plausible scenario in which the C-lobe of CaM remains tethered to NtMKP1, and the N-lobe is free to recruit a second target protein to the complex, such as an NtMKP1 target. Thus, we hypothesize that CaM may be capable of functioning as a Ca²⁺-dependent adaptor or recruiter protein.Key Words: calmodulin, calcium, EF-hand, adaptor protein, mitogen-activated protein kinase phosphataseCalcium (Ca²⁺) is a dynamic secondary messenger that regulates many signaling events in both plant and animal cells. Intracellular Ca²⁺ transients and oscillations (Ca²⁺ signals) are decoded by a large superfamily of calcium-binding proteins, the most important of which is calmodulin (CaM).^1–3 The prototypical CaM protein consists of four tandem helix-loop-helix “EF-hand” Ca²⁺-binding motifs that are divided into distinct N- and C-terminal globular lobes connected by a flexible linker. CaM proteins from all species including the single mammalian CaM and the many different plant CaM isoforms each undergo similar Ca²⁺-induced conformational changes involving a rearrangement of the position of its α-helices that opens distinct hydrophobic target protein-binding patches on the surface of each lobe; known as the “open” conformation (Fig. 1B). These hydrophobic patches can interact with numerous different target proteins including protein kinases, protein phosphatases, cytoskeletal proteins and other cell signaling enzymes, to regulate their activity. The closed or semi-open conformations adopted by the N- and C-lobes of Ca²⁺-free CaM (apo-CaM) (Fig. 1A) can also interact with another subset of proteins, to target CaM to certain cellular locations or facilitate Ca²⁺-independent regulatory events.^1–3Open in a separate windowFigure 1Structures of CaM and CaM-target complexes. (A) apo-CaM (PDB:1DMo), (B) Ca²⁺-CaM (PDB:1CLL). Complexes of CaM bound to (C) CaMBD of smooth muscle myosin light chain kinase (PDB:1CDL), (D) partial CaMBD of plasma membrane Ca²⁺-pump C20W (PDB:1CFF), (E) the adenylyl cyclase protein from Bacillus anthracis (PDB:1K93), (F) 2 glutamate decarboxylase CaMBD''s (PDB:1NWD), (G) 2 CaM proteins bound to 2 small conductance Ca²⁺-activated potassium channel (SK channel) CaMBD''s (PDB:1G4Y), (H) 2 apo-CaM proteins bound to 2 tandem IQ motifs from murine myosin V (PDB:2IX7). In each panel CaM is shown in ivory, the target molecule is shown in blue and the Ca²⁺ ions bound to the N- and/or C-lobes of CaM are represented by red spheres.The CaM-dependent regulation of target proteins can occur through numerous different mechanisms. For example, Ca²⁺-CaM can relieve autoinhibition by binding to a short (20–25 residue) calmodulin-binding domain (CaMBD) sequence that is adjacent to or within an autoinhibitory region of the enzyme (Fig. 2A).³ Numerous structures of these Ca²⁺-CaM-CaMBD complexes have been reported, which reveal a characteristic “wrap-around” binding mode (Fig. 1C). Typically the CaM C-lobe binds with high affinity to a Trp residue within the N-terminal part of the target sequence, and the flexible central linker allows the N-lobe to pivot and bind to a second bulky hydrophobic “anchor” residue within the C-terminal part of the target sequence.³ Truncation of this second anchor residue can lead to binding of only one CaM domain and an extended CaM conformation (Fig. 1D).^4,5 Studies with plant CaM isoforms having mutations to non-CaMBD-coordinating residues have also suggested that a secondary binding interface exists on the opposite surface of the CaM protein which also contributes to the activation of some of these target enzymes.^6,7Open in a separate windowFigure 2Schematic model for the various mechanisms of CaM-dependent target regulation. (A) autoinhibitory domain displacement, (B) sequestering of a ligand binding site, (C) active-site reorganization, (D) CaM-induced target protein dimerization (1:2 complex), (E) CaM-induced target protein dimerization (2:2 complex), (F) hypothesized model for CaM acting as an adaptor/recruiter protein. In each panel CaM is shown as a red dumbbell shaped molecule with Ca²⁺ ions represented by yellow circles, and the target proteins are shown in various colors. See the text for details on each model.Another regulatory mechanism involving Ca²⁺-CaM-binding to a single contiguous CaMBD sequence may occur with the potato kinesin-like CaM-binding protein (KCBP)⁸ as well as some plant cyclic-nucleotide gated channels (CNGC''s).⁹ In both cases the Ca²⁺-CaM binding site on the target protein overlaps with the respective ligand binding site, and thus the binding of KCBP to microtubules or the binding of cyclic nucleotide monophosphates to CNGC''s may be prevented by interaction with Ca²⁺-CaM (Fig. 2B). In a variation on this mechanism, CaM can bind to the cytoplasmic juxtamembrane region of the human epidermal growth factor receptor and sequester a threonine residue which is a specific phosphorylation target of protein kinase C (PKC). CaM-binding inhibits PKC phosphorylation of this threonine, and PKC phosphorylation inhibits CaM-binding.¹⁰There are also several examples of CaM-target interactions where the N- and C-lobes bind to noncontiguous target protein regions, and play distinct roles in target regulation. The structures of a CaM-activated adenylyl cyclase from Bacillus anthracis with and without bound CaM shows how the N- and C-lobes of CaM can bind two distant regions of the adenylyl cyclase enzyme and induce a conformation reorganization that creates the enzyme''s active site (Figs. 1E and ​and2C2C).¹¹ An interesting feature of this interaction is that the CaM N-lobe remains Ca²⁺-free and in a closed conformation, while the C-lobe is in a canonical Ca²⁺-bound open conformation. Indeed, Ca²⁺-binding to the C-lobe but not N-lobe is required for activation of the adenylyl cyclase.¹²The N- and C-lobes of Ca²⁺-CaM can also each simultaneously bind to identical peptides derived from the petunia glutamate decarboxylase (GAD) enzyme to form a 1:2 Ca²⁺-CaM:GAD complex (Fig. 1F).^13,14 This suggests that Ca²⁺-CaM-induced target protein dimerization may be another way in which CaM can regulate target proteins (Fig. 2D). CaM-dependent dimerization has also been shown to regulate the activity of small conductance Ca²⁺-activated K⁺ channels (SK channel), although in this case a novel 2:2 CaM:SK channel complex is formed (Figs. 1G and ​and2E2E).¹⁵ This structure is also unique because Ca²⁺ is bound to the “lower affinity” N-lobe EF-hands, but not to the “higher affinity” C-lobe EF-hands of CaM.In addition to the SK channel, CaM can regulate voltage-gated sodium channels, voltage-gated calcium channels, as well as ryanodine-sensitive calcium release channels.¹⁶ With these channels CaM typically binds in complex Ca²⁺-dependent and Ca²⁺-independent ways to several noncontiguous target sequences in the same protein, and often to so-called IQ motifs (IQXXXRGXXXR). IQ motifs are generally thought to be constitutive apo-CaM binding sites which retain CaM under resting (low [Ca²⁺]) cellular conditions to ensure a rapid response to Ca²⁺-stimuli.¹⁷ However many IQ motifs can also bind specifically to Ca²⁺-CaM or to both apo-CaM and Ca²⁺-CaM. Structures of some Ca²⁺-CaM-IQ domain complexes have revealed wrap-around binding modes, albeit with differences in lobe and peptide orientation compared to other complexes.^18–20 For a discussion about the mechanisms of CaM-dependent ion channel regulation (see ref. ¹⁶). A very recent crystal structure of apo-CaM bound to an IQ domain from myosin V (Fig. 1H) has also revealed yet another variation on the wrap-around binding mode, where the apo-C-lobe of CaM adopts a semi-open conformation and forms numerous interactions with the target sequence, while the apo-N-lobe adopts a closed conformation and forms weaker interactions with the IQ domain.²¹Using several biophysical techniques we recently characterized the interaction between CaM isoforms (mammalian CaM, soybean CaM isoforms SCaM-1 and SCaM-4) and a novel CaMBD derived from the Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1).²² The NtMKP1 protein was initially identified as a CaM-binding protein by Ohashi and coworkers,²³ and the same group recently showed that CaM-binding NtMKP1 homologs are also present in other plant species as well.²⁴ We found that each CaM isoform was capable of binding to the NtMKP1 CaMBD in the absence of Ca²⁺ using only the apo-C-lobe, with the primary binding site consisting of NtMKP1 residues N438 - S449, and additional C-terminal residues G450 - K460 enhancing the overall binding affinity (K_d ∼10⁻⁵ M). In the presence of Ca²⁺, a 1:1 complex could be formed with the CaM C-lobe having significantly increased affinity for the N438 - S449 region of NtMKP1 (K_d 10⁻⁷ − 10⁻¹⁰ M). However, the Ca²⁺-loaded CaM N-lobe interacted only very weakly with the C-terminal NtMKP1 sequence in this 1:1 complex, despite an abundance of seemingly suitable hydrophobic “anchor” residues in this region. Interestingly, the addition of more peptide triggered the independent binding of a second NtMKP1 peptide to the Ca²⁺-CaM N-lobe (Kd 10⁻⁵ − 10⁻⁶ M) to form a 1:2 Ca²⁺-CaM:NtMKP1 complex. As with GAD, these results suggest that CaM is capable of facilitating the dimerization of NtMKP1, although the independent and sequential NtMKP1 peptide binding to the C- and N-lobes markedly distinguishes the CaM-NtMKP1 interaction from the simultaneous high-affinity binding of 2 GAD CaMBD''s to CaM.While our NtMKP1 study was ongoing, Ohashi and coworkers reported that CaM is incapable of stimulating the phosphatase activity of the NtMKP1 enzyme, thereby implying that the CaM-NtMKP1 interaction is necessary for something other than direct enzyme regulation.²⁵ The independent and sequential binding of the NtMKP1 fragments to the Ca²⁺-saturated C- and then N-lobes of CaM observed in our study suggests a plausible situation in which the C-lobe of CaM is tightly bound to NtMKP1, leaving the N-lobe free to recruit a different target protein to the complex, for example, a NtMKP1 protein substrate. Therefore, CaM may be capable of acting as an adaptor or recruiter protein, which would add yet another mechanism of target regulation to CaM''s repertoire (Fig. 2F). In addition to NtMKP1 peptides, the isolated N-lobe of CaM is capable of binding to other CaMBD peptides^26,27 as well as intact target proteins,²⁸ increasing the likelihood that the N-lobe could serve as a recruiter domain. The pre-association of the apo-C-lobe of CaM with NtMKP1 under resting conditions would also ensure a rapid response response to Ca²⁺-stimuli, since CaM would only need to recruit one rather than both protein targets.Although the ability of CaM to act as an adaptor protein in vivo has not yet been demonstrated, there are examples of related EF-hand proteins acting as adaptor proteins, including centrin²⁹ and calcium- and integrin-binding protein 1.³⁰ With the abundance of poorly characterized CaM-binding proteins in plants, many of which have CaMBD''s with little sequence resemblance to the better characterized motifs in animals¹ it seems likely that sequences will be identified which bind preferentially to the CaM N-lobe. Considering the incredible assortment of known CaM interaction modes and regulatory mechanisms, many of which have only been identified within the last decade, it is likely only a matter of time before CaM is proven to function as an adaptor protein in vivo.     

CPK13, a Noncanonical Ca2+-Dependent Protein Kinase,Specifically Inhibits KAT2 and KAT1 Shaker K+ Channels and Reduces Stomatal Opening

Ca²⁺-dependent protein kinases (CPKs) form a large family of 34 genes in Arabidopsis (Arabidopsis thaliana). Based on their dependence on Ca²⁺, CPKs can be sorted into three types: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially calcium-insensitive CPKs. Here, we report on the third type of CPK, CPK13, which is expressed in guard cells but whose role is still unknown. We confirm the expression of CPK13 in Arabidopsis guard cells, and we show that its overexpression inhibits light-induced stomatal opening. We combine several approaches to identify a guard cell-expressed target. We provide evidence that CPK13 (1) specifically phosphorylates peptide arrays featuring Arabidopsis K⁺ Channel KAT2 and KAT1 polypeptides, (2) inhibits KAT2 and/or KAT1 when expressed in Xenopus laevis oocytes, and (3) closely interacts in plant cells with KAT2 channels (Förster resonance energy transfer-fluorescence lifetime imaging microscopy). We propose that CPK13 reduces stomatal aperture through its inhibition of the guard cell-expressed KAT2 and KAT1 channels.Stomata are microscopic organs at the leaf surface, each made of two so-called guard cells forming a pore. Opening or closing these pores is the way through which plants control their gas exchanges with the atmosphere (i.e. carbon dioxide uptake to feed the photosynthetic process and transpirational loss of water vapor). Stomatal movements result from osmotically driven fluxes of water, which follow massive exchanges of solutes, including K⁺ ions, between the guard cells and the surrounding tissues (Hetherington, 2001; Nilson and Assmann, 2007).Both Ca²⁺-dependent and Ca²⁺-independent signaling pathways are known to control stomatal movements (MacRobbie, 1993, 1998; Blatt, 2000; Webb et al., 2001; Mustilli et al., 2002; Israelsson et al., 2006; Marten et al., 2007; Laanemets et al., 2013). In particular, Ca²⁺ signals have been reported to promote stomatal closure through the inhibition of inward K⁺ channels and the activation of anion channels (Blatt, 1991, 1992, 2000; Thiel et al., 1992; Grabov and Blatt, 1999; Schroeder et al., 2001; Hetherington and Brownlee, 2004; Mori et al., 2006; Marten et al., 2007; Geiger et al., 2010; Brandt et al., 2012; Scherzer et al., 2012). However, little is known about the molecular identity of the links between Ca²⁺ events and Shaker K⁺ channel activity. Several kinases and phosphatases are believed to be involved in both the Ca²⁺-dependent and Ca²⁺-independent signaling pathways. Plants express two large kinase families whose activity is related to Ca²⁺ signaling. Firstly, CBL-interacting protein kinases (CIPKs; 25 genes in Arabidopsis [Arabidopsis thaliana]) are indirectly controlled by their interaction with a set of calcium sensors, the calcineurin B-like proteins (CBLs; 10 genes in Arabidopsis). This complex forms a fascinating network of potential Ca²⁺ signaling decoders (Luan, 2009; Weinl and Kudla, 2009), which have been addressed in numerous reports (Xu et al., 2006; Hu et al., 2009; Batistic et al., 2010; Held et al., 2011; Chen et al., 2013). In particular, some CBL-CIPK pairs have been shown to regulate Shaker channels such as Arabidopsis K⁺ Transporter1 (AKT1; Xu et al., 2006; Lan et al., 2011) or AKT2 (Held et al., 2011). Second, Ca²⁺-dependent protein kinases (CPKs) form an even larger family (34 genes in Arabidopsis) of proteins combining a kinase domain with the ability to bind Ca²⁺, thanks to the so-called EF hands (Harmon et al., 2000; Harper et al., 2004). CPKs, which, interestingly, are not found in animal cells, exhibit different calcium dependencies (Boudsocq et al., 2012). With respect to this, three types of CPKs can be considered: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially Ca²⁺-insensitive CPKs (however, structurally close to kinases of groups 1 and 2).Pioneering work by Luan et al. (1993) demonstrated in Vicia faba guard cells that inward K⁺ channels were regulated by some Ca²⁺-dependent kinases. Then, such a Ca²⁺-dependent kinase was purified from guard cell protoplasts of V. faba and shown to actually phosphorylate the in vitro-translated KAT1 protein, a Shaker channel subunit natively expressed in Arabidopsis guard cells (Li et al., 1998). KAT1 regulation by CPK was shown by the inhibition of KAT1 currents after the coexpression of KAT1 and CDPK from soybean (Glycine max) in oocytes (Berkowitz et al., 2000). Since then, several cpk mutant lines of Arabidopsis have been shown to be impaired in stomatal movements, for example cpk10 (Ca²⁺ insensitive), cpk4/cpk11 (Ca²⁺ dependent), and cpk3/cpk6/cpk23 (Ca²⁺ dependent; Mori et al., 2006; Geiger et al., 2010; Munemasa et al., 2011; Hubbard et al., 2012).Of the nine genes encoding voltage-dependent K⁺ channels (Shaker) in Arabidopsis (Véry and Sentenac, 2002, 2003; Lebaudy et al., 2007; Hedrich, 2012), six are expressed in guard cells and play a role in stomatal movements: the Gated Outwardly-Rectifying K⁺ (GORK) gene, encoding an outward K⁺ channel subunit, and the AKT1, AKT2, Arabidopsis K⁺ Rectifying Channel1 (AtKC1), KAT1, and KAT2 genes, encoding inward K⁺ channel subunits (Pilot et al., 2001; Szyroki et al., 2001; Hosy et al., 2003; Pandey et al., 2007; Lebaudy et al., 2008a). Shaker channels result from the assembly of four subunits, and it has been shown that inward subunits tend to heterotetramerize, thus potentially widening the functional and regulatory scope of inward K⁺ conductance in guard cells (Xicluna et al., 2007; Jeanguenin et al., 2008; Lebaudy et al., 2008a, 2010). Inhibition of inward K⁺ channels has been shown to reduce stomatal opening (Liu et al., 2000; Kwak et al., 2001). This has grounded a strategy for disrupting inward K⁺ channel conductance in guard cells by expressing a nonfunctional KAT2 subunit (dominant negative mutation) in a kat2 knockout Arabidopsis line. The resulting Arabidopsis lines, named kincless, have no functional inward K⁺ channels and exhibit delayed stomatal opening (Lebaudy et al., 2008b) with, in the long term, a biomass reduction compared with the Arabidopsis wild-type line.Among the CPKs presumably expressed in Arabidopsis guard cells (Leonhardt et al., 2004), we looked for CPK13, which belongs to the atypical Ca²⁺-insensitive type of CPKs (Kanchiswamy et al., 2010; Boudsocq et al., 2012; Liese and Romeis, 2013) and whose role remains unknown in stomatal movements. Here, we confirm first that CPK13 kinase activity is independent of Ca²⁺ and show that CPK13 expression is predominant in Arabidopsis guard cells using CPK13-GUS lines. We then report that overexpression of CPK13 in Arabidopsis induces a dramatic default in stomatal aperture. Based on the previously reported kincless phenotype (Lebaudy et al., 2008b), we propose that CPK13 could reduce the activity of inward K⁺ channels in guard cells, particularly that of KAT2. We confirm this hypothesis by voltage-clamp experiments and show an inhibition of KAT2 and KAT1 activity by CPK13 (but not that of AKT2). In addition, we present peptide array phosphorylation assays showing that CPK13 targets, with some specificity, several KAT2 and KAT1 polypeptides. Finally, we demonstrate that KAT2 and CPK13 interact in planta using Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM).     

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

Protein 14-3-3zeta binds to protein phosphatase PP1gamma2 in bovine epididymal spermatozoa

The protein phosphatase PP1gamma2 is critical in the regulation of sperm motility and fertility. Its activity is regulated by its binding proteins and by phosphorylation. We have recently shown that PP1gamma2 is phosphorylated and that the amount of phosphorylated PP1gamma2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439-447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1gamma2 during column chromatography of bovine sperm extracts. Western blot analyses confirmed the presence of protein 14-3-3 not only in bovine spermatozoa but also in spermatozoa of diverse species-bull, hamster, horseshoe crab, monkey, rat, turkey, and Xenopus. The binding between PP1gamma2 and protein 14-3-3 was confirmed by coimmunoprecipitation experiments and in pull-down assays with recombinant GST-14-3-3. Western blot analysis and protein 14-3-3 immunoprecipitates with antibodies against the consensus binding domain of protein 14-3-3 reveal that, in addition to PP1gamma2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1gamma2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of bovine spermatozoa. Together, these results provide conclusive evidence that protein 14-3-3 is present in mature spermatozoa and that PP1gamma2 is one of its binding partners.     

Protein kinase C alpha modulates the Ca2+ influx phase of the Ca2+ response to 1alpha,25-dihydroxy-vitamin-D3 in skeletal muscle cells.

Treatment of chick skeletal muscle cells with 1alpha,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] triggers a rapid and sustained increase in cytosolic Ca2+ ([Ca2+]i), which depends on Ca2+ mobilization from inner stores and extracellular Ca2+ entry. Fluorimetric analysis of changes in [Ca2+]i in Fura-2-loaded cells revealed that the hormone significantly stimulates the Ca2+ influx phase within the concentration range of 10(-12)-10(-6) M, with maximal effects (3.5-fold increase) at 10(-9) M 1alpha,25(OH)2D3. The effects of the sterol on the Ca2+ entry pathway were abolished by the PKC inhibitors bisindolylmaleimide and calphostin. We have recently shown that, in these cells, 1alpha,25(OH)2D3 activates and translocates PKC alpha to the membrane, suggesting that this isozyme accounts for PKC-dependent 1alpha,25(OH)2D3 modulation of Ca2+ entry. The role of PKC alpha was specifically addressed here using antisense technology. When the expression of PKC alpha was selectively knocked out by intranuclear microinjection of an antisense oligonucleotide against PKC alpha mRNA, the Ca2+ influx component of the response to 1alpha,25(OH)2D3 was markedly reduced (-60%). These results demonstrate that 1alpha,25(OH)2D3-induced activation of PKC alpha enhances extracellular Ca2+ entry partially contributing to maintainance of the sustained phase of the Ca2+ response to the sterol.     

THE IN VIVO BIOSYNTHESIS OF CHOLESTEROL FROM (3RS,2R) [2-14C,2-3H]MEVALONIC ACID IN THE BRAINS OF RATS ON NORMAL AND PROTEIN DEFICIENT DIETS.1 THE STEREOCHEMISTRY OF TRITIUM ATOMS AT C-7 AND C-15

Abstract— 3RS.2R) [2-¹⁴C,2-³H]Mevalonic acid was injected into the midline thalamus section of the brain of 13-day old rats born and nursed by mothers fed isocaloric diets containing 8% and 25% casein. After 4.5 h. the rats were killed, the brains removed, and the biosynthesized cholesterols were isolated. It was determined that the overall outcome of biosynthetic events at C-7 and C-15 in cholesterol biosynthesized in the rat liver homogenates and in the midline thalamus of the brain of rats raised on the 8% and 25% casein diets is the same.