Ca2+-calmodulin-dependent protein kinase II potentiates store-operated Ca2+ current

A rise in intracellular Ca2+ (Ca2+i) mediates various cellular functions ranging from fertilization to gene expression. A ubiquitous Ca2+ influx pathway that contributes significantly to the generation of Ca2+i signals, especially in non-excitable cells, is store-operated Ca2+ entry (SOCE). Consequently, the modulation of SOCE current affects Ca2+i dynamics and thus the ensuing cellular response. Therefore, it is important to define the mechanisms that regulate SOCE. Here we show that a rise in Ca2+i potentiates SOCE. This potentiation is mediated by Ca2+-calmodulin-dependent protein kinase II (CaMKII), because inhibition of endogenous CaMKII activity abrogates Ca2+i-mediated SOCE potentiation and expression of constitutively active CaMKII potentiates SOCE current independently of Ca2+i. Moreover, we present evidence that CaMKII potentiates SOCE by altering SOCE channel gating. The regulation of SOCE by CaMKII defines a novel modulatory mechanism of SOCE with important physiological consequences.     

Expression and purification of a convenient Ca2+-calmodulin-dependent protein kinase II GST-fusion substrate

Abundant and convenient protein substrates are extremely useful tools for studying protein kinases. However, few such substrates exist for alpha-Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and those that are available are generally small and expensive peptides that are cumbersome to use. The GST-fusion expression system was used to express a 10 amino acid substrate of CaMKII PLRRTLSVAA in bacteria. Using glutathione-agarose affinity chromatography, we obtained milligram quantities of the highly purified recombinant GST-fusion protein. The GST-fusion protein was tested for its efficacy and specificity as a substrate for CaMKII in phosphorylation assays using recombinant enzyme and radiolabeled [gamma-32P]ATP. The reaction products of these phosphorylation assays were resolved by electrophoresis in SDS-polyacrylamide gels and quantified by phosphoimage analysis. It was found that compared to a phosphorylation-null substrate, GST-PLRRTLAVAA, in which the phosphorylated target serine residue was mutated to an alanine, the GST-PLRRTLSVAA substrate was phosphorylated by CaMKII with an apparent K(m) of 18 microM, indicating that the latter is a highly effective substrate for this enzyme.     

Ca2+-calmodulin-dependent regulation of F-actin-myelin basic protein interaction

Myelin basic proteins (MBP) interacts with F-actin resulting in the precipitation of a complex of both proteins. Electron microscope observations of this complex reveal the presence of ordered bundles of F-actin filaments similar to those obtained from F-actin and troponin I. In addition to the bundles, there also appear short fragments of F-actin filaments. In the presence of Ca2+ calmodulin causes a release of MBP from its complex with F-actin, accompanied by dissociation of F-actin bundles into separate filaments. Parallel to the binding of MBP to F-actin the ATPase activity of actomyosin is progressively reduced. This inhibition is reversed by calmodulin but only in the presence of Ca2+. Studies of the binding of S-1 to F-actin and to the F-actin-MBP complex indicate that the interaction sites for MBP and S-1 on the actin molecule are different.     

Ca2+/calmodulin-dependent protein kinase II inhibitors disrupt AKAP79-dependent PKC signaling to GluA1 AMPA receptors

GluA1 (formerly GluR1) AMPA receptor subunit phosphorylation at Ser-831 is an early biochemical marker for long-term potentiation and learning. This site is a substrate for Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and protein kinase C (PKC). By directing PKC to GluA1, A-kinase anchoring protein 79 (AKAP79) facilitates Ser-831 phosphorylation and makes PKC a more potent regulator of GluA1 than CaMKII. PKC and CaM bind to residues 31-52 of AKAP79 in a competitive manner. Here, we demonstrate that common CaMKII inhibitors alter PKC and CaM interactions with AKAP79(31-52). Most notably, the classical CaMKII inhibitors KN-93 and KN-62 potently enhanced the association of CaM to AKAP79(31-52) in the absence (apoCaM) but not the presence of Ca(2+). In contrast, apoCaM association to AKAP79(31-52) was unaffected by the control compound KN-92 or a mechanistically distinct CaMKII inhibitor (CaMKIINtide). In vitro studies demonstrated that KN-62 and KN-93, but not the other compounds, led to apoCaM-dependent displacement of PKC from AKAP79(31-52). In the absence of CaMKII activation, complementary cellular studies revealed that KN-62 and KN-93, but not KN-92 or CaMKIINtide, inhibited PKC-mediated phosphorylation of GluA1 in hippocampal neurons as well as AKAP79-dependent PKC-mediated augmentation of recombinant GluA1 currents. Buffering cellular CaM attenuated the ability of KN-62 and KN-93 to inhibit AKAP79-anchored PKC regulation of GluA1. Therefore, by favoring apoCaM binding to AKAP79, KN-62 and KN-93 derail the ability of AKAP79 to efficiently recruit PKC for regulation of GluA1. Thus, AKAP79 endows PKC with a pharmacological profile that overlaps with CaMKII.     

Evidence that Ser-82 is a unique phosphorylation site on vimentin for Ca2(+)-calmodulin-dependent protein kinase II.

We identified the sites on vimentin that are phosphorylated by Ca2(+)-calmodulin-dependent protein kinase II (CaM-kinase II). Sequential analysis of the purified phosphopeptides demonstrated that the sites are -Thr-Arg-Thr-Tyr-Ser(PO4)38-Leu-Gly-Ser-Ala- and -Val-Arg-Leu-Leu-Gln-Asp-Ser(PO4)82-Val-Asp-, which are located within the amino-terminal head domain of vimentin. For Ser-82 but not Ser-38, the proposed CaM-kinase II recognition amino acid sequence (Arg-X-X-Ser/Thr) was not found. Studies with a series of synthetic peptide analogs corresponding to Ser-82 and its surrounding amino acid sequence indicate that Asp-84 acts as an essential substrate specificity determinant for the Ser-82 phosphorylation by CaM-kinase II. The CaM-kinase II recognition site may be more extensive than heretofore determined.     

Ca2(+)-calmodulin-dependent protein kinase II phosphorylates various types of non-epithelial intermediate filament proteins

We have investigated the actions of Ca2(+)-calmodulin (CaM)-dependent protein kinase II on various types of non-epithelial intermediate filament proteins, vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament triplet proteins. Most of these filament proteins could serve as substrates. The effects of phosphorylation on the filamentous structure of vimentin were investigated in sedimentation experiments and by using electron microscopy. The amount of unassembled vimentin increased linearly with increased phosphorylation. However, the extent of the effect of phosphorylation on the potential to polymerize was also affected by the MgCl2 concentration, under conditions for reassembly. The actions of Ca2(+)-CaM-dependent protein kinase II on non-epithelial intermediate filaments under physiological conditions are given attention.     

Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase.

The cmd1-6 allele contains three mutations that block Ca2+ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone alpha-factor. The 50% lethal dose (LD50) of alpha-factor for the calmodulin mutant is almost fivefold below the LD50 for a wild-type strain. The calmodulin mutants are not more sensitive to alpha-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca2+-calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD50 of alpha-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to alpha-factor. Activation of the Ca2+-calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD50 of alpha-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.     

Ca2+-calmodulin-dependent polymerization of actin by myelin basic protein

The interaction between myelin basic protein (MBP) and G-actin was studied under nonpolymerizing conditions, i.e.,2mM HEPES, pH 7.5, 0.1 mM CaCl2 and 0.2 mM ATP. Fluorescence studies using pyrenyl-actin and the measurements of ATP hydrolysis rate show that MBP induces changes in the structure of the actin monomer similar to those occurring during polymerization by salt. Electron microscope observations of the MBP-G-actin complex reveal the presence of filamentous structures which appear as separate filaments or as bundles of filaments in lateral association. These filaments are polar as visualized by attachment of heavy meromyosin. The biochemical data together with electron microscope observations suggest that the binding of MBP to G-actin under non-polymerizing conditions induces an interaction between actin monomers leading to the formation of filamentous structures which may be similar to F-actin filaments. The effects of MBP on G-actin can be reversed by calmodulin in the presence of Ca2+.     

Allosteric regulation of Ca2+-calmodulin-dependent phosphodiesterase activity from the bovine brain

The Ca2+-calmodulin-dependent interaction of phosphodiesterase with phenyl-Sepharose was demonstrated. BSA caused incomplete competitive inhibition of phosphodiesterase activation by calmodulin. The 17-fold increase of the constant for phosphodiesterase activation by calmodulin was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis; in this case the value of the inhibition constant amounted to Ki approximately 6 microM. In the absence of calmodulin saturating concentrations of BSA reduced the enzyme activity nearly 3-4-fold. The effect of BSA on phosphodiesterase was incompetitive with respect to cAMP (Ki approximately 1.4 microM). Both phenomena are characteristic of incompetitive binding of BSA to the enzyme with respect to cAMP and calmodulin. Gel filtration data reflect the changes in the enzyme molecular weight during its interaction with BSA. All the above reactions of the enzyme are reversible.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Pressure overload selectively up-regulates Ca2+/calmodulin-dependent protein kinase II in vivo

Signals transduced by the multifunctional calcium/calmodulin-dependent protein kinases (CaMKs), have been suggested to regulate the development of hypertrophy. We address the role of the three multifunctional CaMKs, CaMK I, II, and IV, in this process using transverse aortic constriction (TAC) to induce cardiac hypertrophy in mice. We find a 33% increase in total CaMK activity 7 d after TAC. However, there are no changes in the levels of CaMKI, which is expressed in the ventricles, or CaMKIV, which is not detectable in the ventricles. Moreover, mice null for the CaMKIV gene develop ventricular hypertrophy and induce the expression of selected hypertrophy marker mRNAs, indicating that CaMKIV is not required at any time during the development of hypertrophy. On the other hand, TAC does increase both mRNA and protein levels of specific isoforms of CaMKII derived from both gamma and delta genes. Included among these isoforms are those that localize to both cytoplasm and nucleus. Collectively, the increased levels of CaMKII isoforms result in a constitutive increase in the Ca(2+)/calmodulin-independent activity of CaMKII in the ventricles. We conclude that CaMKII is the multifunctional CaMK most likely to mediate Ca(2+)- dependent protein phosphorylation events in response to TAC-induced cardiac hypertrophy.     

Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein

The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.     

The phosphorylation site of Ca2+-dependent protein kinase from alfalfa

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca²⁺-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.     

Endothelial cell sodium-potassium-chloride cotransport. Evidence of regulation by Ca2+ and protein kinase C

Previous studies have shown that vascular endothelial cells exhibit a highly active Na-K-Cl cotransport system that is regulated by a variety of vasoactive hormones and neurotransmitters, suggesting that the cotransporter may play an important role in endothelial cell function. In this study, the regulation of endothelial cell Na-K-Cl cotransport was further investigated by probing the stimulus-transfer pathway by which vasoactive agents stimulate the cotransporter. Specifically, three peptides previously shown to stimulate cotransport activity (angiotensin II, vasopressin, and bradykinin) were evaluated. Na-K-Cl cotransport was assessed in cultured bovine aortic endothelial cells as bumetanide-sensitive K+ influx. Stimulation of Na-K-Cl cotransport by angiotensin II, vasopressin, or bradykinin was found to be reduced either by removal of extracellular Ca2+ or by treatment of the cells with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In addition, the calmodulin antagonist W-7 was found to prevent stimulation of endothelial cell Na-K-Cl cotransport by the three peptides. These findings suggest that regulation of endothelial cell cotransport by these vasoactive peptides may be both Ca(2+)- and calmodulin-dependent. Angiotensin II, vasopressin, and bradykinin were also found to elevate phosphatidylinositol hydrolysis in the cultured endothelial cells. Thus, the possibility that regulation of endothelial Na-K-Cl cotransport by these vasoactive peptides also involves diacylglycerol activation of protein kinase C was investigated. A 10-min exposure of the endothelial cells to low doses of phorbol 12-myristate 13-acetate was found to reduce Na-K-Cl cotransport whether in the presence or absence of angiotensin II, vasopressin, or bradykinin. However, down-regulation of protein kinase C by a 40-h exposure to higher doses of the phorbol ester was found to elevate Na-K-Cl cotransport activity under both control and agonist-stimulated conditions, indicating that activation of protein kinase C results in inhibition of endothelial cell Na-K-Cl cotransport. Thus, protein kinase C activation may serve as negative feedback in the stimulus-transfer pathway by which these agonists regulate endothelial cell Na-K-Cl cotransport.     

Reduced Ca2+-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure

Studies on the status of multifunctional Ca(2+)-calmodulin (CaM)-dependent protein kinase-II (CaMKII) in failing hearts are limited and controversial. The study was performed in the left ventricular (LV) myocardium of six dogs with heart failure (HF) (LV ejection fraction, 23 +/- 2%) and six normal (NL) dogs. In the LV homogenate, CaMKII activity and its protein level were determined by using the CaMKII peptide and antibody, respectively. Furthermore, the protein level of CaM and phosphorylated phospholamban (PLB) at threonine-17 (PLB-Thr(17)) and serine-16 (PLB-Ser(16)) were also determined in the LV homogenate using a specific antibody. In addition, the level of zinc, which inhibits protein kinase A activity, was determined in the LV tissue by inductively coupled plasma mass spectrometry. CaMKII activity and phosphorylated PLB-Thr(17) and PLB-Ser(16) levels, but not CaM and Zn levels, were significantly reduced in the LV homogenate of dogs with HF compared with NL dogs. These results suggest that CaMKII activity is reduced in the failing LV myocardium, and this abnormality is associated with reduced protein expression level of the enzyme but not due to changes in CaM and zinc levels. In conclusion, reduced CaMKII activity and phosphorylated PLB level may be partly responsible for impaired sarcoplasmic reticulum function in HF.     

Spermatogenesis and the regulation of Ca(2+)-calmodulin-dependent protein kinase IV localization are not dependent on calspermin

Calspermin and Ca(2+)-calmodulin-dependent protein kinase IV (CaMKIV) are two proteins encoded by the Camk4 gene. CaMKIV is found in multiple tissues, including brain, thymus, and testis, while calspermin is restricted to the testis. In the mouse testis, both proteins are expressed within elongating spermatids. We have recently shown that deletion of CaMKIV has no effect on calspermin expression but does impair spermiogenesis by disrupting the exchange of sperm basic nuclear proteins. The function of calspermin within the testis is unclear, although it has been speculated to play a role in binding and sequestering calmodulin during the development of the germ cell. To investigate the contribution of calspermin to spermatogenesis, we have used Cre/lox technology to specifically delete calspermin, while leaving kinase expression intact. We unexpectedly found that calspermin is not required for male fertility. We further demonstrate that CaMKIV expression and localization are unaffected by the absence of calspermin and that calspermin does not colocalize to the nuclear matrix with CaMKIV.