Functional processing of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N): evidence for a critical role of proteolytic processing in the regulation of its catalytic activity, subcellular localization and substrate targeting in vivo

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, the proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo.     

Phosphorylation and activation of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca2+/calmodulin-dependent protein kinase I (CaMKI)

Nuclear Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.     

Identification and characterization of CaMKP-N, nuclear calmodulin-dependent protein kinase phosphatase.

Calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and concomitantly deactivates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs), such as CaMKI, CaMKII, and CaMKIV. In the present study, a nuclear CaMKP-related protein, CaMKP-N, was identified. This protein consisted of 757 amino acid residues with a calculated molecular weight of 84,176. Recombinant CaMKP-N dephosphorylated CaMKIV. The activity of CaMKP-N requires Mn(2+) ions and is stimulated by polycations. Transiently expressed CaMKP-N in COS-7 cells was localized in the nucleus. This finding together with previous reports regarding localization of CaMKs indicates that CaMKP-N dephosphorylates CaMKIV and nuclear CaMKII, whereas CaMKP dephosphorylates CaMKI and cytosolic CaMKII.     

Identification and characterization of nuclear localization signals of CaMKP-N

Calmodulin-dependent protein kinase phosphatase (CaMKP) and CaMKP-N dephosphorylate and regulate multifunctional Ca(2+)/calmodulin-dependent protein kinases. The enzymatic properties of CaMKP-N and CaMKP resemble each other, whereas their localizations are different. CaMKP-N is localized in the nucleus, whereas CaMKP is localized in the cytosol. In the present study, the nuclear localization signals (NLSs) of CaMKP-N were identified and characterized. CaMKP-N contains two NLSs, NLS1 and NLS2, at the C-terminus. A cluster of basic residues in the NLSs is important for their function. NLS1 and NLS2 function independently, but mutagenesis analysis suggests that these NLSs interact with each other.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II by expression of mutated cDNAs in Escherichia coli.

The cDNAs encoding the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were ligated into the bacterial expression vector pET and expressed in Escherichia coli. The bacterially expressed alpha and beta subunits exhibited Ca2+/calmodulin-dependent activity and were easily purified to apparent homogeneity from cell extracts. To determine the minimum size required for catalytic activity and the properties of the calmodulin-binding domain, mutated CaM kinase II cDNAs were expressed in E. coli and the enzymatic property of expressed proteins was examined. The replacement of Thr-286 of the alpha subunit with the negatively charged amino acid Asp or that of Arg-283 with the neutral amino acid Gly induced the partially Ca2+ independent activity. The mutant enzymes alpha-I(delta 283-478) and alpha-II(delta 359-478), which truncated the C-terminal region of the alpha subunit, exhibited CaM kinase II activity and the activities of alpha-I(delta 283-478) and alpha-II(delta 359-478) were completely independent of and partially dependent on Ca2+ and calmodulin, respectively. However, the truncated protein alpha(delta 250-478), which was only 33 amino acids shorter than the alpha-I(delta 283-478) protein had no enzymatic activity, indicating that alpha-I(delta 283-478) was close to the minimum size of the active form. The mutant enzyme alpha(delta 291-315), which lacked the calmodulin-binding domain exhibited Ca2+ independent activity. The molecular mass was, however, smaller than that expected from the amino acid sequence. The mutant enzyme alpha(delta 304-315), which lacked the C-terminal half of the calmodulin-binding domain of the alpha subunit, however, exhibited Ca(2+)-independent activity without a reduction in molecular size, indicating that residues 304-315 of the alpha subunit constituted the core calmodulin-binding domain.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the alpha subunit

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg much less than Lys much less than Gln much less than Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase alpha subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2(+)-independent kinase activity (5.46 +/- 0.88%) compared to the wild-type Arg283 (1.86 +/- 0.71%) and the Gln283 mutant (2.15 +/- 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2(+)-independent activity, the Arg283 kinase attained maximal Ca2(+)-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2(+)-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2(+)-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.     

Nature and Immunochemical Characteristics of a Ca2+/Calmodulin Kinase Activity Endowed in a Highly Insoluble Protein Purified from Adult Rat Brain

Using sequential extraction procedure of proteins from adult rat forebrain, a protein of Mr 52,000, insoluble in neutral detergents, capable of binding calmodulin in the presence of Ca2+, was isolated. Antibodies to this antigen had the capacity to inhibit the Ca2+/calmodulin-dependent kinase activity associated with this protein. This protein (52K) (in many respects identical to the major protein of postsynaptic densities) shares by itself the Ca2+/calmodulin-dependent kinase activity, thus differing from soluble Ca2+/calmodulin-dependent kinases isolated by others. Despite its insolubility in most detergents, the 52K protein is not particularly rich in hydrophobic amino acids. Its richness in cysteine and proline residues suggests that the active conformation of the enzyme is sustained by numerous disulfide bridges.     

Knockdown of two splice variants of Ca(2+)/calmodulin-dependent protein kinase Iδ causes developmental abnormalities in zebrafish, Danio rerio

We isolated cDNA clones for zebrafish Ca(2+)/calmodulin-dependent protein kinase I (zCaMKI) δ isoforms by expression screening using cDNA library from embryos at 72-h post-fertilization (hpf). There are two splice variants with different C-terminal sequences, comprising of 392 and 368 amino acids, and they are designated zCaMKIδ-L (long form) and zCaMKIδ-S (short form), respectively. Although recombinant zCaMKIδ-L and zCaMKIδ-S expressed in Escherichia coli showed essentially the same catalytic properties including substrate specificities, they showed different spatial and temporal expression. Western blotting analysis using the isoform-specific antibodies revealed that zCaMKIδ-L clearly appeared from 36hpf but zCaMKIδ-S began to appear at 60hpf and thereafter. zCaMKIδ-S was predominantly expressed in brain, while zCaMKIδ-L was widely distributed in brain, eye, ovary and especially abundantly expressed in skeletal muscle. The gene knockdown of zCaMKIδ using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. Severe phenotype of embryos exhibited short trunk, kinked tail and small heads. These phenotypes could be rescued by coinjection with the recombinant zCaMKIδ, but not with the kinase-dead mutant. These results clearly indicate that the kinase activity of zCaMKIδ plays a crucial role in the early stages in the embryogenesis of zebrafish.     

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells. Identification of a novel neuronal calmodulin-dependent protein kinase

Ca2+-sensitive protein kinases are thought to play a pivotal role in Ca2+-mediated neuronal communication. We describe here the cloning, purification, and characterization of a major Ca2+/calmodulin-dependent, brain-specific protein kinase which is particularly enriched in cerebellar granule cells. The enzyme is comprised of Mr 65,000 and 67,000 polypeptides which copurify to homogeneity and phosphorylate synapsin I. The protein kinase is coded for by two poly(A+) RNAs of 2.0 and 3.5 kilobases which probably derive from a single gene. Two cDNA inserts, one of 198 base pairs and one of 1225 base pairs, contain a total of 677 base pairs of the protein coding sequence which includes sequences homologous to other calmodulin-dependent protein kinases including part of the calmodulin-binding domain. The surprising presence of extended sequences which are enriched in glutamate residues may influence the subcellular distribution of this kinase. Immunohistochemical localization with an affinity-purified antibody reveals that whereas the enzyme is expressed in several neuronal subpopulations, it is exceptionally enriched in the granule cells of the cerebellum. The relevance of the biochemical, molecular, and histologic properties of this enzyme is discussed in the context of neuronal Ca2+ signaling.     

Ca/calmodulin-dependent protein kinase phosphatase (CaMKP) is indispensable for normal embryogenesis in zebrafish, Danio rerio

Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and regulates multifunctional Ca²⁺/calmodulin-dependent protein kinases (CaMKs). However, the biological functions of this enzyme have not been clarified in vivo. To investigate the biological significance of CaMKP during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP (zCaMKP). The isolated cDNA clone possessed an open reading frame of 1272 bp encoding 424 amino acids and shared 47% and 48% amino acid identity with rat and human CaMKP, respectively. Interestingly, zCaMKP lacks the Glu cluster corresponding to residues 101-109 in the rat enzyme, and was not activated by polycations such as poly-l-lysine. The recombinant zCaMKP required Mg²⁺ rather than Mn²⁺ for activity. Furthermore, zCaMKP dephosphorylated CaMKIV but not phosphorylase a, α-casein, or extracellular signal-regulating kinase (ERK), suggesting that the enzyme regulates Ca²⁺ signaling pathways in zebrafish. Cotransfection of zCaMKP with mammalian CaMKI significantly decreased phospho-CaMKI in ionomycin-stimulated 293T cells. During embryogenesis, the expression of zCaMKP increased gradually after 48 h post-fertilization, as demonstrated by Western blotting using an anti-zCaMKP antibody. The knockdown of the zCaMKP gene with morpholino-based antisense oligonucleotides resulted in an increased incidence of embryos with severe morphological and cellular abnormalities, i.e., a significant increase in the number of round-shaped embryos and apoptotic cells in the whole body. A marked decrease in zCaMKP expression was observed in the antisense- but not control oligo-injected embryos. Embryonic death was rescued by coinjection with recombinant rat CaMKP but not with phosphatase-dead mutant (D194A). These results clearly show the significance of zCaMKP during zebrafish embryogenesis.     

Phosphorylation of smooth muscle caldesmon by three protein kinases: implication for domain mapping

Phosphorylation of duck gizzard caldesmon by Ca2+/phospholipid-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase and casein kinase II has been investigated. The Ca2+/phospholipid-dependent protein kinase incorporates more than 3 mol phosphate per mol (140 kDa) caldesmon. All phosphorylation sites are localized in the actin- and calmodulin-binding peptide (40-45 kDa) supposed to be a part of the C-terminal domain of caldesmon. Casein kinase II phosphorylates only one site located in a short (25-27 kDa) peptide, presumably in the caldesmon N-terminal domain. The Ca2+/calmodulin-dependent protein kinase phosphorylates two sites located in the N- and C-terminal domains of caldesmon.     

Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation

Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase converts it from a Ca2(+)-dependent to a Ca2(+)-independent or autonomous kinase, a process that may underlie some long-term enhancement of transient Ca2+ signals. We demonstrate that the neuronal alpha subunit clone expressed in COS-7 cells (alpha-CaM kinase) is sufficient to encode the regulatory phenomena characteristic of the multisubunit kinase isolated from brain. Activity of alpha-CaM kinase is highly dependent on Ca2+/calmodulin. It is converted by autophosphorylation to an enzyme capable of Ca2(+)-independent (autonomous) substrate phosphorylation and autophosphorylation. Using site-directed mutagenesis, we separately eliminate five putative autophosphorylation sites within the regulatory domain and directly examine their individual roles. Ca2+/calmodulin-dependent kinase activity is fully retained by each mutant, but Thr286 is unique among the sites in being indispensable for generation of an autonomous kinase.     

Phosphorylation of lactate dehydrogenase by protein kinases

The influence of phosphorylation on the properties of lactate dehydrogenase (LDH) has been studied. Data obtained using the immobilization approach support the assumption that the autophosphorylation of LDH discovered previously in the presence of ATP has no relation to protein kinase activity of the enzyme. Phosphorylation of native LDH by tyrosine kinases was shown to be inefficient. However, the efficiency of the phosphorylation considerably increased after the dissociation of LDH into non-native forms of the enzyme. Ca2+/calmodulin-dependent protein kinase catalyzes incorporation of 0.8-0.9 mole phosphate per mole of LDH tetramer. The phosphorylation results in an increase in activity by 25-30% and increases markedly the stability of the enzyme during cold inactivation. Phosphorylation of LDH by Ca2+/calmodulin-dependent protein kinase, unlike the phosphorylation on tyrosine residues, is supposed to be of importance for the control of cell metabolism.     

C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin

Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.     

Phosphorylation of the elongation factor 2: the fifth Ca2+/calmodulin-dependent system of protein phosphorylation

Elongation factor 2 (EF-2) has been recently shown to be extensively phosphorylated in a Ca2+/calmodulin-dependent manner in extracts of mammalian cells (A. G. Ryazanov (1987) FEBS Lett. 214, 331-334). In the present study, we partially purified the protein kinase which phosphorylates EF-2 from rabbit reticulocytes. The molecular weight of the enzyme determined by gel filtration was about 140,000. Unlike the substrate, the EF-2 kinase had no affinity for RNA and therefore could be separated from EF-2 by chromatography on RNA-Sepharose. After chromatography on hydroxyapatite, the kinase activity became calmodulin-dependent. Two-dimensional separation of the phosphorylated EF-2 according to O'Farrell's technique revealed that there were two phosphorylation sites within the EF-2 molecule; in both cases, the phosphorylated amino acid was threonine. The EF-2 kinase differed from the four known types of Ca2+/calmodulin-dependent protein kinases. Thus, the system of EF-2 phosphorylation represents the novel (fifth) Ca2+/calmodulin-dependent system of protein phosphorylation. This system is supposed to be responsible for the regulation of the elongation rate of protein biosynthesis in eukaryotic cells.     

CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells

Ca(2+)-regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) is highly expressed in exocrine glands and was shown to regulate digestive enzyme secretion from pancreatic acinar cells. We found CRHSP-28 highly expressed in cultured mucosal secretory T84 cells, consistent with an important regulatory role in apical membrane trafficking. Stimulation of cells with carbachol (CCh) induced rapid, concentration-dependent phosphorylation of CRHSP-28 on at least two serine residues. Isoelectric focusing and immunoblotting were used to characterize cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation depends on elevated cellular Ca2+, being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTAAM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, phosphopeptide mapping studies demonstrated that CaMKII induced an identical phosphopeptide profile to endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated phosphorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated that CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaMKII was also localized to the subapical cytoplasm and was clearly displaced following actin filament disruption. These data suggest that CRHSP-28 phosphorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells.     

Evidence for the activation of the multifunctional Ca2+/calmodulin-dependent protein kinase in response to hormones that increase intracellular Ca2+

The phosphorylation state of six cytoplasmic proteins is increased following treatment of isolated rat hepatocytes with hormones that elevate free intracellular Ca2+ levels (Garrison, J. C. and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Tryptic 32P-phosphopeptide maps of two of the substrates, pyruvate kinase and a 49,000-dalton protein, the major 32P-labeled protein in hepatocytes, were prepared following stimulation of cells with vasopressin, a Ca2+-linked hormone. Peptide maps of the 49,000-dalton protein phosphorylated in vitro with the recently identified multifunctional Ca2+/calmodulin-dependent protein kinase contained phosphopeptides identical to those observed in the intact cell, suggesting that this kinase is activated in response to Ca2+-mobilizing hormones. Similar in vitro phosphorylation experiments with pyruvate kinase suggested that the Ca2+/calmodulin-dependent protein kinase can phosphorylate not only the serine residues observed following vasopressin stimulation of the intact cell but also additional threonine residues. Both pyruvate kinase and the 49,000-dalton protein are also phosphorylated in the hepatocyte in response to glucagon and in vitro by the cAMP-dependent protein kinase. Both vasopressin and glucagon appear to stimulate the phosphorylation of identical serine residues in pyruvate kinase but only vasopressin enhances the phosphorylation of certain sites in the 49,000-dalton protein. Comparison of the tryptic phosphopeptide maps of these substrates phosphorylated in vitro with either the Ca2+/calmodulin-dependent protein kinase or the cAMP-dependent protein kinase suggests that the Ca2+-dependent kinase can phosphorylate unique sites in both substrates. It appears to share specificity at other sites with the cAMP-dependent protein kinase. Overall, the results suggest that the multifunctional Ca2+/calmodulin-dependent protein kinase plays an important role in the response of the hepatocyte to a Ca2+ signal.     

A brain-specific Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca2+ signaling.

A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.     

Calmodulin kinase II chimeras used to investigate the structural requirements for smooth muscle myosin light chain kinase autoinhibition and calmodulin-dependent activation

Segments of the autoregulatory domain of MK, a catalytically active fragment of the monomeric smooth muscle myosin light chain kinase (smMLCK) (residues 472-972), were replaced with their counterparts from a homologous but multimeric enzyme, calmodulin-dependent protein kinase II (CaM KII). Chimeric proteins in which both the autoregulatory and oligomerization domains of CaM KII (residues 281-478) were substituted for residues 781-972 of smMLCK, MK(CK281-478), or only the autoregulatory domain of CaM KII (residues 281-315) was exchanged for residues 781-813 of smMLCK, MK(CK281-315), exhibited significant enzymatic activity in the absence of Ca(2+)/CaM. In contrast, both MK and a chimeric protein in which the C-terminal half of the autoregulatory domain of smMLCK was replaced with CaM KII residues 301-315, MK(CK301-315), were inactive in the absence of Ca(2+)/CaM. These results indicate that the sequence of the N-terminal half of the autoregulatory domain of smMLCK is important for complete autoinhibition of its enzymatic activity. All proteins bound to Ca(2+)/CaM, and the chimeric proteins MK(CK281-478) and MK(CK281-315) were activated by Ca(2+)/CaM with activation constants (K(CaM)) and maximal enzymatic activities comparable to those of the wild-type MK enzyme. This demonstrates that the entire autoregulatory domain of CaM KII can replace that of smMLCK in its ability to promote efficient CaM-dependent activation of the smMLCK enzyme. However, the inability of the chimeric protein MK(CK301-315) to be activated by Ca(2+)/CaM suggests that replacement of only the C-terminal half of the autoregulatory domain of smMLCK, while still retaining the ability to bind Ca(2+)/CaM, also substitutes residues that prevent activation of the enzyme by Ca(2+)/CaM.     

Ca2+/calmodulin-dependent protein kinase II isoenzymes gamma and delta are both present in H+/K+-ATPase-containing rabbit gastric tubulovesicles.

Ca2+/calmodulin-dependent protein kinase II is thought to participate in M3 muscarinic receptor-mediated acid secretion in gastric parietal cells. During acid secretion tubulovesicles carrying H+/K+-ATPase fuse with the apical membrane. We localized Ca2+/calmodulin-dependent protein kinase II from highly purified rabbit gastric tubulovesicles using Ca2+/calmodulin-dependent protein kinase II isoform-specific antibodies, in vitro phosphorylation and pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II activity by the potent Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. The presence of Ca2+/calmodulin-dependent protein kinase II in tubulovesicles was shown by immunoblot detection of both Ca2+/calmodulin-dependent protein kinase II-gamma (54 kDa) and Ca2+/calmodulin-dependent protein kinase II-delta (56.5 kDa). The immunoprecipitated Ca2+/calmodulin-dependent protein kinase II from tubulovesicles showed Ca2+/calmodulin-dependent protein kinase activity by phosphorylating autocamtide-II, a specific synthetic Ca2+/calmodulin-dependent protein kinase II substrate. KN-62 inhibited the in vitro autophosphorylation of tubulovesicle-associated Ca2+/calmodulin-dependent protein kinase II (IC50 = 11 nM). During the search for potential Ca2+/calmodulin-dependent protein kinase II substrates we identified different proteins associated with tubulovesicles, such as synaptophysin and beta-tubulin immunoreactivity, which were identified using specific antibodies. These targets are known to participate in intracellular membrane traffic. Ca2+/calmodulin-dependent protein kinase II is thought to play an important role in regulating tubulovesicular motor activity and therefore in acid secretion.