Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.     

Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.     

Identification of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II in the postsynaptic density by protein sequencing and mass spectrometry.

Previously we detected more than 28 PSD proteins to be phosphorylated by CaM kinase II, and identified 14 protein substrates (Yoshimura, Y., Aoi, T., Yamauchi, T., Mol. Brain Res. 81, 118-128, 2000). In the present study, the remaining substrates were analyzed by protein sequencing and mass spectrometry. We found 6 proteins not previously known to be substrates of CaM kinase II, namely PSD95-associated protein, SAP97, TOAD-64, TNF receptor-associated protein, insulin-receptor tyrosine kinase 58/53 kDa substrate, and homer 1b.     

Cloning,characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin-dependent protein kinase I gamma in the rat brain

Ca2+/calmodulin-dependent protein kinase I (CaMKI), originally identified as a protein kinase phosphorylating synapsin I, has been shown to constitute a family of closely related isoforms (alpha, beta and gamma). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKI gamma 1 and -gamma 2. CaMKI gamma 1 and -gamma 2 contain an identical N-terminal catalytic domain with different C-terminal regions due to the deletion of the 425-bp nucleotide sequence of CaMKI gamma 1 in CaMKI gamma 2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+/CaM-dependent activity of CaMKI gamma 1 by the preincubation with Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), but no significant activation of CaMKI gamma 2. Northern blot analysis has demonstrated the predominant expression of CaMKI gamma in the brain. RT-PCR analysis has revealed similar expression patterns between CaMKI gamma 1 and CaMKI gamma 2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKI gamma mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108-15 cells, transfected CaMKI gamma 1 and -gamma 2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKI gamma may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.     

Auto-inhibition of Ca(2+)/calmodulin-dependent protein kinase II by its ATP-binding domain

Ca(2+)/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca(2+)/CaM binds to it. In this inactive form CaMPK-II does not bind ATP suggesting that the ATP-binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP-binding domain (CaMPK-II(23-34), GAFSVVRRCVKV) can inhibit the Ca(2+)/CaM-dependent activity (IC(50) of 3 microM) but has no effect on the Ca(2+)/CaM-independent activity of CaMPK-II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide-2 and ATP. The inhibition by F12 showed specificity towards CaMPK-II, but also inhibited CaMPK-I (IC(50) = 12.5 microM), while CaMPK-IV (IC(50) = 85 microM) was inhibited poorly and cAMP-dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca(2+)/CaM-dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP-binding domain appears to play a role in the regulation of nonphosphorylated CaMPK-II activity.     

14-3-3 Proteins directly regulate Ca(2+)/calmodulin-dependent protein kinase kinase alpha through phosphorylation-dependent multisite binding

Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha) plays critical roles in the modulation of neuronal cell survival as well as many other cellular activities. Here we show that 14-3-3 proteins directly regulate CaMKKalpha when the enzyme is phosphorylated by protein kinase A on either Ser74 or Ser475. Mutational analysis revealed that these two serines are both functional: the CaMKKalpha mutant with a mutation at either of these residues, but not the double mutant, was inhibited significantly by 14-3-3. The mode of regulation described herein differs the recently described mode of 14-3-3 regulation of CaMKKalpha.     

Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.     

Dual effect of ATP in the activation mechanism of brain Ca(2+)/calmodulin-dependent protein kinase II by Ca(2+)/calmodulin.

The activation mechanism of Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal [T?r?k, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820] is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin [Drum et al. (2000) J. Biol. Chem. 275, 36334-36340] (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca(2+)/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alphaCaMKII as well as with alphaCaMKII-derived calmodulin-binding peptides in the presence of Ca(2+). These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alphaCaMKII with estimated interprobe distances of 40 and 22-26 A. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca(2+)/TA-cal with alphaCaMKII is estimated as 2 x 10(7) M(-1) s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca(2+)/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M(-1) s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: II. Mutational analysis

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Cloning of a Ca(2+)/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides

A Ca(2+)/calmodulin-dependent protein kinase (CaMK) gene was cloned and characterized from Arthrobotrys dactyloides, a nematode-trapping fungus. The resulting 373-amino-acid protein, FCaMK, has significant homology to mammalian CaMKs. FCaMK contains a serine/threonine kinase domain followed by a calmodulin-binding domain. The activation loop in FCaMK (amino acids 184-199) contains a phosphorylation site at threonine-188, which could be the target of a kinase activator. Truncated FCaMK mutants revealed that amino acids 296-324 are essential for calmodulin binding. An oligopeptide designed from residues 297-324 formed a stable peptide-calmodulin complex of 1:1 stoichiometry. Southern blot analysis detected a single copy of the fcamk gene, suggesting that FCaMK plays an important role in Ca(2+)/calmodulin signaling in A. dactyloides.     

Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca/calmodulin-dependent protein kinase II from rat brain

Multifunctional Ca²⁺/calmodulin-dependent protein kinase type II (CaMK II) plays a crucial role in mediation of cellular responses to rising cytosolic Ca²⁺ levels. We find that the novel peptide substrate PGTIEKKRSNAMKKMKSIEQHR serves as a highly potent substrate for CaMK II enzymes purified from both Drosophila and rat. The peptide is derived from a photoreceptor-specific protein, phosrestin I, of the Drosophila compound eye and is designated as phosrestide-1. Using saturating substrate concentrations, the enzymes from both species transfer the γ-phosphoryl group of ATP to phosrestide-1 at a level three to ten times greater than to the commercially available mammalian-derived CaMK II substrates, autocamtide-3 and syntide-2. This indicates a conservation of substrate preferences for CaMK II derived from distantly related species, a dipteran fly and a mammal. Although phosrestide-1 contains two potential serine residues for CaMK II phosphorylation, we find that only the C-terminal serine is phosphorylated by rat CaMK II. However, removal of the upstream sequence containing the N-terminal serine substantially reduced the potency of phosrestide-1 as a CaMK II substrate to a level comparable to that of syntide-2 or autocamtide-3. We also find that a peptide representing the N-terminal segment of phosrestide-1 does not inhibit either CaMK II. Therefore, the enhanced potency of phosrestide-1 as a CaMK II substrate is likely to be due to a preferred conformation of the peptide induced by the N-terminal segment rather than to a specific binding of the enzymes to the N-terminus of the peptide. To the best of our knowledge, phosrestide-1 is the first CaMK II substrate which is designed based on an invertebrate sequence. The high phosphorylation level of phosrestide-1 by CaMK II of mammalian origin may reflect highly conserved CaMK II signaling cascades between vertebrates and invertebrates.     

Extracellular calcium induces activation of Ca(2+)/calmodulin-dependent protein kinase II and mediates spontaneous activation in rat oocytes

Ovulated rat oocytes are activated spontaneously soon after recovery from the oviducts. To investigate the kinetics and mechanism of rat oocyte spontaneous activation (OSA), we investigated the effect of aging in oviducts, hyaluronidase treatment, and extracellular and intracellular calcium, and examined the activity of CaMKII and the effect of its inhibitor on OSA. Oocyte aging in oviducts and hyaluronidase did not affect OSA. However, OSA was significantly decreased in calcium-free medium and in calcium-containing medium containing L-type calcium channel blocker and IP(3)R inhibitor. Moreover, significantly lower OSA was shown with an inhibitor of CaMKII. There was a significant increase of CaMKII activity at 30min after oocyte recovery and constitutively active CaMKII was located near the meiotic spindle in freshly recovered oocytes. Therefore, CaMKII is one of the upstream signals to activate rat oocytes spontaneously after recovery and rat oocytes respond very sensitively to extracellular calcium.     

Investigation of neuronal cell type-specific gene expression of Ca2+/calmodulin-dependent protein kinase II

The promoter activity of the rat Ca²⁺/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5′-flanking region of α and β isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5′-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types. Published: April 12, 2002     

Up-regulation of a non-kinase activity isoform of Ca(2+)/calmodulin-dependent protein kinase kinase beta1 (CaMKKbeta1) in hibernating bat brain

To improve hybrid tilapia (Oreochromis niloticusxO. aureus) survival under cold shock, the influence of diets containing various dietary lipids was investigated. Four different diets were used which consisted of 12% fish oil, 12% palmitoleic oil 12% coconut oil, and a mixture of fish oil (7%) and corn oil (5%). Our results showed that during cold shock, the proportion of saturated fatty acids in the fish steadily and significantly decreased for all of the diets, but the proportion of monounsaturated fatty acids increased. Proportions of polyenoic fatty acids initially increased then stabilized for the mixed, fish, and coconut oil diets, but did not significantly increase until day 4 for the palmitoleic oil diet. The stearoyl-CoA desaturase (SCD) activity was the lowest on day 0 and then gradually increased for all diets. At any point, the enzymatic activity of SCD was the highest for fish on the mixed and the coconut oil diet, followed by the palmitoleic oil diet, and was lowest for the fish oil diet. The expression of SCD mRNA steadily increased for all diets, but increased more substantially for the mixed diet. On day 6, the expression was the highest for fish on the mixed diet, followed by the coconut oil diet, with the lowest levels for those on the palmitoleic and fish oil diets. These results show that dietary lipids strongly affect the fatty acid composition and SCD expression in tilapia under cold shock, and cold tolerance of this species is also affected.     

Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle

The relation between CaM kinase II activity and high Ca²⁺-mediated stress responses was studied in cultured vascular smooth muscle cells. Treatment with ionomycin (1 M) for 5 min caused a significant loss of CaM kinase II activity in whole cell homegenates and prominent vesiculation of the endoplasmic reticulum (ER). Similar losses of CaM kinase II activity were observed in the soluble lysate as assessed by activity measurements and Western blotting. Examination of the post-lysate particulate fraction showed that the loss of CaM kinase II from the soluble lysate was accompanied by a redistribution of CaM kinase II to this fraction. The ionomycin-mediated response was limited to this concentration (1 M); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 M) or ATP (100 M) did not cause a shift in CaM kinase II distribution. Treatment with neither the CaM kinase II inhibitor KN-93 nor the phosphatase inhibitor okadaic acid altered the ionomycin-induced redistribution indicating that CaM kinase II activation and/or phosphorylation was not part of the mechanism. The response, however, was eliminated when the cells were treated in Ca²⁺-free medium. Washout of ionomycin led to only a partial restoration of the kinase activity in the soluble fraction after 10 min. Immunofluorescence microscopy of resting cells indicated colocalization of antibodies to CaM kinase II and an ER protein marker. ER vesiculation induced by ionomycin coincided with a parallel redistribution of CaM kinase II and ER marker proteins. These data link ionomycin-induced ER restructuring to a progressive redistribution of CaM kinase II protein to an insoluble particulate fraction and loss of cellular CaM kinase II activity. We propose that redistribution of CaM kinase II and loss of cellular activity are components of a common Ca²⁺-overload induced cellular stress response in cells.     

Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization

Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.