Mechanism of autophosphorylation of the multifunctional Ca2+/calmodulin-dependent protein kinase

The multifunctional Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol undergoes a self-phosphorylation or autophosphorylation reaction. Our conclusion that this reaction is autocatalytic is based on the following lines of evidence: The autophosphorylation reaction and the protein kinase activity toward other substrates are absolutely dependent on the presence of both Ca2+ and calmodulin; autophosphorylation and phosvitin kinase activity show a similar time course and indistinguishable heat lability; the reaction is a consistent property of every preparation of rat brain kinase; the reaction is present in both crude and highly purified preparations of similar kinases or isozymes from rat lung, spleen, heart, bovine brain, and a neuronal tissue from Aplysia californica, a marine mollusk; phosphorylation of the kinase subunits is not mimicked by addition of cAMP, cGMP, Ca2+ plus diglyceride, or addition of the cAMP-dependent protein kinase, and is not blocked by the heat-stable inhibitor protein of the cAMP-dependent protein kinase; and the reaction is intramolecular. Autophosphorylation results in the stoichiometric incorporation of phosphate into both the 51,000- and 60,000-dalton subunits.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin-independent autophosphorylation

The autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) results in the generation of kinase activity that is largely Ca2+/CaM-independent. We report that continued Ca2+/CaM-independent autophosphorylation of CaM-KII results in the generation of distinct phosphopeptides as identified by high performance liquid chromatography and enzymatic properties that are different than those observed for Ca2+/CaM-dependent autophosphorylation. These Ca2+/CaM-independent properties include (a) increased catalytic activity, (b) higher substrate affinity for the phosphorylation of synapsin I, and (c) decreased CaM-binding to both CaM-KII subunits as analyzed by gel overlays. Our results indicate that the autophosphorylation of only one subunit per holoenzyme is required to generate the Ca2+/CaM-independent CaM-KII. We suggest a two-step process by which autophosphorylation regulates CaM-KII. Step I requires Ca2+/CaM and underlies initial kinase activation. Step II involves continued autophosphorylation of the Ca2+/CaM-independent kinase and results in increased affinity for its substrate synapsin I and decreased affinity for calmodulin. These results indicate a complex mechanism through which autophosphorylation of CaM-KII may regulate its activity in response to transient fluctuations in intracellular calcium.     

Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.

Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase.     

Phosphorylation and activation of Ca2+/calmodulin-dependent protein kinase phosphatase by Ca2+/calmodulin-dependent protein kinase II.

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKPase) is a protein phosphatase which dephosphorylates autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) and deactivates the enzyme (Ishida, A., Kameshita, I. and Fujisawa, H. (1998) J. Biol. Chem. 273, 1904-1910). In this study, a phosphorylation-dephosphorylation relationship between CaMKII and CaMKPase was examined. CaMKPase was not significantly phosphorylated by CaMKII under the standard phosphorylation conditions but was phosphorylated in the presence of poly-L-lysine, which is a potent activator of CaMKPase. The maximal extent of the phosphorylation was about 1 mol of phosphate per mol of the enzyme and the phosphorylation resulted in an about 2-fold increase in the enzyme activity. Thus, the activity of CaMKPase appears to be regulated through phosphorylation by its target enzyme, CaMKII.     

Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells.

We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells.     

Regulation of human CLC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase

The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation.     

Multifunctional Ca2+/calmodulin-dependent protein kinase

Multifunctional Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) is a prominent mediator of neurotransmitters which elevate Ca²⁺. It coordinates cellular responses to external stimuli by phosphorylating proteins involved in neurotransmitter synthesis, neurotransmitter release, carbohydrate metabolism, ion flux and neuronal plasticity. Structure/function studies of CaM kinase have provided insights into how it decodes Ca²⁺ signals. The kinase is kept relatively inactive in its basal state by the presence of an autoinhibitory domain. Binding of Ca²⁺/calmodulin eliminates this inhibitory constraint and allows the kinase to phosphorylate its substrates, as well as itself. This autophosphorylation significantly slows dissociation of calmodulin, thereby trapping calmodulin even when Ca²⁺ levels are subthreshold. The kinase may respond particularly wel to multiple Ca²⁺ spikes since trapping may enable a spike frequency-dependent recruitment of calmodulin with each successive Ca²⁺ spike leading to increased activation of the kinase. Once calmodulin dissociates, CaM kinase remains partially active until it is dephosphorylated, providing for an additional period in which its response to brief Ca²⁺ transients is potentiated.Special issue dedicated to Dr. Paul Greengard.     

Phosphorylation of smooth myosin light chain kinase by smooth muscle Ca2+/calmodulin-dependent multifunctional protein kinase

Smooth muscle myosin light chain kinase (MLC kinase) was phosphorylated by smooth muscle calmodulin-dependent protein kinase II (CaM protein kinase II). When MLC kinase was free from calmodulin, two sites were phosphorylated. The phosphorylation at the one site was much faster than the other site; however, the phosphorylation at the first site was completely blocked by calmodulin binding to MLC kinase. Phosphorylation of MLC kinase by CaM protein kinase II increased the dissociation constant of MLC kinase for calmodulin about 10 times without changing the Vmax. The location of the phosphorylation sites was identified by isolating and sequencing the tryptic phosphopeptides of MLC kinase. The preferred site was identified as serine 512 and the second site as serine 525. These sites are the same as the sites phosphorylated by cAMP-dependent protein kinase.     

Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase

To search for the downstream target protein kinases of Ca (2+)/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKalpha catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5'-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr (189) in the activation loop. Phosphorylation of Thr (189) by CaMKKalpha induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr (189) phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKalpha (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKalpha is capable of activating SAD-B through phosphorylation of Thr (189) both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.     

The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.     

Activation of multifunctional Ca2+/calmodulin-dependent kinase in intact hippocampal slices.

In vitro phosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) converts it to a form that is independent of Ca2+. We demonstrate that significant Ca(2+)-independent CaM kinase activity is present in untreated hippocampal slices. Two manipulations that produce a long-lasting enhancement of neuronal activity in hippocampal slices, elevated extracellular Ca2+ or depolarization with high K+, generate additional Ca(2+)-independent activity. This increase is dependent on extracellular Ca2+ and is correlated with an increased phosphorylation of CaM kinase. In contrast, CaM kinase in posterior pituitary, a brain structure that is not thought to be involved in memory-related processes, is not modulated by depolarization. These results suggest that the Ca(2+)-independent form of CaM kinase may modulate neuronal activity in the hippocampus.     

Regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase

Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alphaCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca(2+)/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440), exhibited enhanced Ca(2+)/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca(2+)/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.     

Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV

Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.     

Ca2+/calmodulin-dependent protein kinase in Saccharomyces cerevisiae

Ca²⁺-dependent chromatography of soluble cytosolic proteins on calmodulin-Sepharose gave a fraction that exhibited Ca²⁺- and calmodulin-dependent phosphorylation of several polypeptides, including 60, 56 and 45 kDa species. At 0.2 μM beef calmodulin the phosphorylation was optimal at 3 μM free Ca²⁺, and at 80 μM free Ca²⁺ it was half-maximal at about 0.1 μM beef calmodulin. It is concluded that the fraction contains calmodulin-dependent protein kinase(s) which is (are) autophosphorylated or associated with substrates.     

Molecular cloning of Ca2+/calmodulin-dependent protein kinase phosphatase.

Calmodulin-dependent protein kinase (CaM-kinase) phosphatase dephosphorylates and concomitantly deactivates CaM-kinase II activated upon autophosphorylation, and CaM-kinases IV and I activated upon phosphorylation by CaM-kinase kinase [Ishida, I., Okuno, S., Kitani, T., Kameshita, I., and Fujisawa, H. (1998) Biochem. Biophys. Res. Commun. 253, 159-163], suggesting that CaM-kinase phosphatase plays important roles in the function of Ca2+ in the cell, because the three multifunctional CaM-kinases (CaM-kinases I, II, and IV) are thought to be the key enzymes in the Ca2+-signaling system. In the present study, cDNA for CaM-kinase phosphatase was cloned from a rat brain cDNA library. The coded protein consisted of 450 amino acids with a molecular weight of 49, 165. Western blot analysis showed the ubiquitous tissue distribution of CaM-kinase phosphatase. Immunocytochemical analysis revealed that CaM-kinase phosphatase is evenly distributed outside the nucleus in a cell.     

Regulation of Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II

The 63-kDa subunit, but not the 60-kDa subunit, of brain calmodulin-dependent cyclic nucleotide phosphodiesterase was phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II. When calmodulin was bound to the phosphodiesterase, 1.33 +/- 0.20 mol of phosphate was incorporated per mol of the 63-kDa subunit within 5 min with no significant effect on enzyme activity. Phosphorylation in the presence of low concentrations of calmodulin resulted in a phosphorylation stoichiometry of 2.11 +/- 0.21 and increased about 6-fold the concentration of calmodulin necessary for half-maximal activation of the phosphodiesterase. Peptide mapping analyses of complete tryptic digests of the 63-kDa subunit revealed two major (P1, P4) and two minor (P2, P3) 32P-peptides. Calmodulin-binding to the phosphodiesterase almost completely inhibited phosphorylation of P1 and P2 with reduced phosphorylation rates of P3 and P4, suggesting the affinity change of the enzyme for calmodulin may be caused by phosphorylation of P1 and/or P2. When Ca2+/calmodulin-dependent protein kinase II was added without prior autophosphorylation, there was no phosphorylation of the 63-kDa phosphodiesterase subunit or of the kinase itself in the presence of a low concentration of calmodulin, and with excess calmodulin the phosphodiesterase subunit was phosphorylated only at P3 and P4. Thus the 63-kDa subunit of phosphodiesterase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II and blocked by Ca2+/calmodulin binding to the subunit.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

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.