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.     

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).     

Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II.

The relationship of the kinase which co-purifies with caldesmon to Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was investigated by studying the phosphorylation of bovine brain synapsin I, as well-characterized substrate of CaM-kinase II. Synapsin I is a very good substrate (Km = 90 nM) of the co-purifying kinase, which phosphorylates two sites in synapsin I, both of which are distinct from the single site phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of synapsin I is Ca2(+)- and calmodulin-dependent: half-maximal activation occurs at 0.13 microM-Ca2+ and maximal activity at 0.4 microM-Ca2+. Phosphorylation of the co-purifying kinase slightly enhances the rate, but does not alter the stoichiometry, of subsequent synapsin I phosphorylation; it does, however, circumvent the requirement for Ca2+ and calmodulin. The properties of this kinase therefore closely resemble those of CaM-kinase II, and we conclude that it is probably a smooth-muscle isoenzyme of CaM-kinase II.     

Glucose-induced stimulation of the Ras-cAMP pathway in yeast leads to multiple phosphorylations and activation of 6-phosphofructo-2-kinase

Yeast cells respond to changes of the environment by complex modifications of the metabolism. An increase of the extracellular glucose concentration activates the Ras-cAMP pathway. Via a production of cAMP this pathway stimulates the cAMP-dependent protein kinase (PKA) which is involved in the posttranslational regulation of the key enzymes of gluconeogenesis and glycolysis. 6-Phosphofructo-2-kinase (PFK2) catalyzes the synthesis of fructose 2,6-bisphosphate, the most potent activator of the glycolytic key enzyme 6-phosphofructo-1-kinase. We investigated the molecular mechanism of the glucose-induced phosphorylation and activation of PFK2 in Saccharomyces cerevisiae. After an incubation of PFK2 with ATP and PKA in vitro, two amino acid residues, Thr157 and Ser644, are phosphorylated and the enzyme is activated. A stimulation of the Ras-cAMP pathway by glucose addition to cultivated yeast cells leads to an in vivo activation of PFK2 which is accompanied by a more complex phosphorylation pattern of the enzyme. The phosphorylation of the protein on Ser644 is the result of PKA stimulation while the protein kinase(s) catalyzing the 5-fold phosphorylation of the peptide fragment T(67)(-)(101) is (are) still unknown. The functional significance of T(67)(-)(101) and its phosphorylation is supported by the finding that PFK2 lacking this peptide is inactive.     

Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.     

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.     

Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia

The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.     

Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein

Transglutaminase 2 (TG2, tissue transglutaminase) is a multifunctional protein involved in cross-linking a variety of proteins, including retinoblastoma protein (Rb). Here we show that Rb is also a substrate for the recently identified serine/threonine kinase activity of TG2 and that TG2 phosphorylates Rb at the critically important Ser780 residue. Furthermore, phosphorylation of Rb by TG2 destabilizes the Rb.E2F1 complex. TG2 phosphorylation of Rb was abrogated by high Ca2+ concentrations, whereas TG2 transamidating activity was inhibited by ATP. TG2 was itself phosphorylated by protein kinase A (PKA). Phosphorylation of TG2 by PKA attenuated its transamidating activity and enhanced its kinase activity. Activation of PKA in mouse embryonic fibroblasts (MEF) with dibutyryl-cAMP enhanced phosphorylation of both TG2 and Rb by a process that was inhibited by the PKA inhibitor H89. Treatment with dibutyryl-cAMP enhanced Rb phosphorylation in MEFtg2+/+ cells but not in MEFtg2-/- cells. These data indicate that Rb is a substrate for TG2 kinase activity and suggest that phosphorylation of Rb, which results from activation of PKA in fibroblasts, is indirect and requires TG2 kinase activity.     

Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C

The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcinerin (Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the Km (from 9.9 +/- 1.1 to 17.5 +/- 1.1 microM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.     

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.     

Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture

Ser55 of neurofilament L (NF-L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP-dependent protein kinase (PKA). Bovine NF-L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin. Trypsin-digested fragments were assigned by MALDI/ TOF (matrix-assisted laser desorption and ionization/ time-of-flight) mass spectrometry. Phosphorylation sites were found at Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site-specific phosphorylation-dependent antibody against Ser55 rendered NF-L phosphorylated at Ser55 detectable in primary cultured rat neurons. One-hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co-treatment with 10 microM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF-L may be phosphorylated at Ser55 by PKA or by a PKA-like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.     

Autophosphorylation of Calmodulin-Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.     

Identification of membrane-bound calcium, calmodulin-dependent protein kinase II in canine heart

Phospholamban, the putative regulatory proteolipid of the Ca2+/Mg2+ ATPase in cardiac sarcoplasmic reticulum, was selectively phosphorylated by a Ca2+/calmodulin (CaM)-dependent protein kinase associated with a cardiac membrane preparation. This kinase also catalyzed the phosphorylation of two exogenous proteins known to be phosphorylated by the multifunctional Ca2+/CaM-dependent protein kinase II (Ca2+/CaM-kinase II), i.e., smooth muscle myosin light chains and glycogen synthase a. The latter protein was phosphorylated at sites previously shown to be phosphorylated by the purified multifunctional Ca2+/CaM-kinase II from liver and brain. The membrane-bound kinase did not phosphorylate phosphorylase b or cardiac myosin light chains, although these proteins were phosphorylated by appropriate, specific calmodulin-dependent protein kinases added exogenously. In addition to phospholamban, several other membrane-associated proteins were phosphorylated in a calmodulin-dependent manner. The principal one exhibited a Mr of approximately 56,000, a value similar to that of the major protein (57,000) in a partially purified preparation of Ca2+/CaM-kinase II from the soluble fraction of canine heart that was autophosphorylated in a calmodulin-dependent manner. These data indicate that the membrane-bound, calmodulin-dependent protein kinase that phosphorylates phospholamban in cardiac membranes is not a specific calmodulin-dependent kinase, but resembles the multifunctional Ca2+/CaM-kinase II. Our data indicate that this kinase may be present in both the particulate and soluble fractions of canine heart.     

Phosphorylation of GRK1 and GRK7 by cAMP-dependent protein kinase attenuates their enzymatic activities

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.     

PTP-PEST: a protein tyrosine phosphatase regulated by serine phosphorylation.

The protein tyrosine phosphatase PTP-PEST is an 88 kDa cytosolic enzyme which is ubiquitously expressed in mammalian tissues. We have expressed PTP-PEST using recombinant baculovirus, and purified the protein essentially to homogeneity in order to investigate phosphorylation as a potential mechanism of regulation of the enzyme. PTP-PEST is phosphorylated in vitro by both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) at two major sites, which we have identified as Ser39 and Ser435. PTP-PEST is also phosphorylated on both Ser39 and Ser435 following treatment of intact HeLa cells with TPA, forskolin or isobutyl methyl xanthine (IBMX). Phosphorylation of Ser39 in vitro decreases the activity of PTP-PEST by reducing its affinity for substrate. In addition, PTP-PEST immunoprecipitated from TPA-treated cells displayed significantly lower PTP activity than enzyme obtained from untreated cells. Our results suggest that both PKC and PKA are capable of phosphorylating, and therefore inhibiting, PTP-PEST in vivo, offering a mechanism whereby signal transduction pathways acting through either PKA or PKC may directly influence cellular processes involving reversible tyrosine phosphorylation.     

Phosphorylation of chicken cardiac C-protein by calcium/calmodulin-dependent protein kinase II.

Chicken cardiac C-protein was readily phosphorylated by purified calcium/calmodulin-dependent protein kinase II (CaM-kinase II). Maximum incorporation was about 4 mol of 32P/mol of C-protein subunit. Peptide mapping indicated that some of the sites phosphorylated by CaM-kinase II were located on the same phosphopeptides obtained when C-protein was phosphorylated by the cAMP-dependent protein kinase (peptides T1, T2, and T3). There was a fourth peptide (T3a) which was unique to CaM-kinase II phosphorylation. 32P-Amino acid analysis showed that essentially all of the 32P of peptides T1, T2, and T3a was in phosphoserine. cAMP-dependent protein kinase incorporated 32P only into threonine of peptide T3. Threonine was the preferred site of phosphorylation by CaM-kinase II, but there was significant phosphorylation of a serine in peptide T3. Partially purified C-protein preparations contained an associated calcium/calmodulin-dependent protein kinase. Peptide maps obtained from C-protein phosphorylated by the endogenous kinase were similar to those obtained from C-protein phosphorylated by CaM-kinase II. However, the ratio of phosphothreonine to phosphoserine in peptide T3 was lower. This was due to a contaminating phosphatase in the partially purified C-protein which preferentially dephosphorylated the phosphothreonine of peptide T3. It is suggested that the calcium/calmodulin-dependent protein kinase associated with C-protein is similar or identical to CaM-kinase II and that CaM-kinase II may play a role in the phosphorylation of C-protein in the heart.     

Identification of phosphorylation sites on murine nuclear lamin C by RP-HPLC and microsequencing

Isolated interphase lamin C, obtained from Ehrlich ascites tumor cells, was digested by Lys-C endoproteinase, the resulting peptides separated by reversed-phase HPLC and subjected to microsequencing in order to identify phosphorylation sites in interphase and following phosphorylation in vitro by cdc2-kinase, protein kinase C (PKC) and protein kinase A (PKA), respectively. Nuclear lamin C showed partial phosphorylation of Ser392 and Ser409, and possibly Ser407 in interphase. Phosphorylation was increased in response to cdc2-kinase at Ser390 and Ser392 and to PKC at Ser572. The N-terminal peptide (aa 1-32) containing consensus sequences for the 3 kinases was phosphorylated by cdc2-kinase, PKC and PKA. The sequence data suggests that multiple molecular switches via lamina modification control the dynamic behaviour of the nucleoskeleton during the cell cycle.     

Phosphorylation and regulation of choline kinase from Saccharomyces cerevisiae by protein kinase A

The CKI1-encoded choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from Saccharomyces cerevisiae was phosphorylated in vivo on multiple serine residues. Activation of protein kinase A activity in vivo resulted in a transient increase in the phosphorylation of choline kinase. This phosphorylation was accompanied by a stimulation in choline kinase activity. In vitro, protein kinase A phosphorylated choline kinase on a serine residue with a stoichiometry (0.44 mol of phosphate/mol of choline kinase) consistent with one phosphorylation site/choline kinase subunit. The major phosphopeptide derived from the enzyme phosphorylated in vitro by protein kinase A was common to one of the major phosphopeptides derived from the enzyme phosphorylated in vivo. Protein kinase A activity was dose- and time-dependent and dependent on the concentrations of ATP (Km 2.1 microM) and choline kinase (Km 0.12 microM). Phosphorylation of choline kinase with protein kinase A resulted in a stimulation (1.9-fold) in choline kinase activity whereas alkaline phosphatase treatment of choline kinase resulted in a 60% decrease in choline kinase activity. The mechanism of the protein kinase A-mediated stimulation in choline kinase activity involved an increase in the apparent Vmax values with respect to ATP (2.6-fold) and choline (2.7-fold). Overall, the results reported here were consistent with the conclusion that choline kinase was regulated by protein kinase A phosphorylation.     

Phosphorylation of the cystic fibrosis transmembrane conductance regulator.

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.