Ascaris suum phosphofructokinase. Phosphorylation by protein kinase and sequence of the phosphopeptide

Phosphorylation of the ascarid phosphofructokinase with the catalytic subunit of beef heart cyclic AMP-dependent protein kinase results in the incorporation of 1 mol of P/mol of subunit. Accompanying the phosphorylation there is a 3-4-fold increase in catalytic activity when measured at pH 6.8 with inhibitory levels of ATP. Studies on the effect of phosphorylation on the ATP saturation curve demonstrated that phosphorylation decreased the inhibitory action of ATP. The apparent Km of the catalytic subunit for the phosphofructokinase was 11.2 microM. Chymotryptic or subtilisin digestion of the labeled enzyme released distinct but overlapping phosphopeptides that were purified by high pressure liquid chromatography and sequenced by gas phase peptide sequencing. The sequence of the chymotryptic peptide was Ala-Lys-Gly-Arg-Ser-Asp-Ser(P)-Ile-Val-Pro-Thr. Based on these results and earlier observations, it is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of energy metabolism in the parasitic helminth.     

Phosphorylation of heart phosphofructokinase by Ca2+/calmodulin protein kinase.

Phosphofructokinase (PFK) from sheep heart was shown to be phosphorylated by Ca2+/calmodulin protein kinase (CaM-kinase) as well as by cyclic AMP-dependent protein kinase (PKA). HPLC analysis of phosphorylated PFK indicated that phosphorylation by CaM-kinase occurs at least at two sites that are distinct from those recognized by PKA. Phosphorylation by either CaM-kinase of PKA resulted in an increase in sensitivity to ATP inhibition and a small but consistent decrease in Ki for ATP. Phosphorylation by either protein kinase caused a slight increase in the Km of PFK for fructose-6-P. Protein kinase C failed to phosphorylate PFK. Combinations of PKA, CaM-kinase and protein kinase C did not alter the stoichiometry of phosphorylation and did not change the effect on enzyme activity.     

Phosphorylation of caldesmon by protein kinase C

Protein kinase C catalyzes phosphorylation of caldesmon, an F-actin binding protein of smooth muscle, in the presence of Ca2+ and phospholipid. Protein kinase C incorporates about 8 mol of phosphate/mol of chicken gizzard caldesmon. When calmodulin was added in the medium, there was an inhibition of phosphorylation. The fully phosphorylated, but not unphosphorylated, caldesmon inhibited myosin light chain kinase activity. The possibility that protein kinase C plays some role in smooth muscle contractile system through caldesmon, warrants further attention.     

Phosphorylation of the cytoskeletal protein talin by protein kinase C

Talin, a component of the focal contact of cultured cells, is an in vitro substrate for protein kinase C. Immunoprecipitation confirms that talin is the phosphorylated protein. Phosphorylation is dependent on both phosphatidylserine and calcium and reaches a level of incorporation of 0.8 mol phosphate/mol protein. Phosphoamino acid analysis demonstrates the presence of phosphoserine and phosphothreonine, but no phosphotyrosine. Two dimensional mapping of tryptic peptides, and V8 peptides reveals the existence of multiple phosphorylation sites. The identification of talin as a substrate for protein kinase C implicates talin as a potential regulator of focal contact organization and perhaps cell morphology.     

Phosphorylation of the calmodulin-dependent protein phosphatase by protein kinase C

The calmodulin-dependent protein phosphatase was shown to be phosphorylated by the Ca2+, phospholipid-dependent protein kinase (protein kinase C). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 61 kDa catalytic subunit was phosphorylated. Phosphorylation by protein kinase C was stimulated up to 15-fold by addition of phosphatidyl-L-serine and between 0.5 to 1.0 mole of phosphate was incorporated per mole of phosphatase. It is possible that protein kinase C is involved in the regulation of the calmodulin-dependent protein phosphatase via this novel phosphorylation of the enzyme.     

Phosphorylation of muscle phosphofructokinase by the catalytic subunit of cyclic AMP-dependent protein kinase.

The catalytic subunit of cyclic AMP-dependent protein kinase catalyzes the phosphorylation of rabbit skeletal muscle phosphofructokinase. The reaction is inhibited by the specific inhibitor of protein kinase and proceeds at about 2% the rate observed with phosphorylase kinase but more rapidly than with rat liver fructose bisphosphatase as substrate. Maximum extent of incorporation (0.43 to 0.85 moles per mole of protomer) plus the covalently-bound phosphate present in the isolated enzyme (0.20 to 0.34 moles per mole) approaches one mole per mole.     

Phosphorylation of laminin-1 by protein kinase C

Several extracellular proteins have been reported to be phosphorylated. Previous studies of our laboratory indicated that laminin-1 can be phosphorylated by protein kinase A (PKA). Moreover, it has been reported that protein kinase C (PKC), although known to be intracellular, can phosphorylate extracellular proteins in the case of cellular damage and/or platelet activation. In the present study we examined the possibility of laminin-1 serving as a substrate of PKC. Amino acid analysis revealed that laminin-1 is phosphorylated by this enzyme on serine residues. Self assembly, heparin binding, and cell attachment on the phosphorylated molecule were then studied. Phosphorylated laminin-1 showed an increased and more rapid self assembly than the non-phosphorylated molecule. Heparin binding and cell attachment experiments indicated enhanced heparin and cell binding capacity of the phosphorylated molecule in comparison to the non- phosphorylated control. These results indicate that laminin-1 can be phosphorylated by protein kinase C. Furthermore, phosphorylation by protein kinase C seems to alter several properties of the molecule, though, the in vivo significance of this phenomenon remains to be studied.     

Phosphorylation of neurofilament proteins by protein kinase C

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional alpha-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [32P]orthophosphate in vivo.     

Phosphorylation by protein kinase CK2 changes the DNA binding properties of the human chromatin protein DEK

We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein. Phosphorylation fluctuates during the cell cycle with a moderate peak during G(1) phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un- or underphosphorylated form of DEK.     

Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Phosphorylation of a chromaffin granule-binding protein by protein kinase C

Protein kinase C was detected in a group of Ca2+-dependent chromaffin granule membrane-binding proteins (chromobindins) on the basis of Ca2+-, phosphatidylserine-, 1,2-diolein-, and phorbol myristate acetate-stimulated histone kinase activity. When the chromobindins were incubated with [gamma-32P]ATP, Ca2+, and phosphatidylserine, 32P was incorporated predominantly into a protein of mass 37 +/- 1 kilodaltons (chromobindin 9, or CB9). Phosphorylation of this protein was also stimulated by diolein and phorbol myristate acetate, indicating that it is a substrate for the protein kinase C activity present in the chromobindins. Maximum phosphate incorporation into CB9 in the presence of 1 mM Ca2+, 75 micrograms/ml of phosphatidylserine, 2.5 micrograms/ml of diolein, and 12.5 micrograms/ml of dithiothreitol was 0.53 mol/mol of CB9 in 5 min. Eight 32P-labeled phosphopeptides were resolved in two-dimensional electrophoretic maps of trypsin digests of CB9. Phosphoamino acid analysis revealed that phosphorylation was exclusively on serine (94%) and threonine (6%) residues. Incubation of the chromobindins with chromaffin granule membranes in the presence of [gamma-32P]ATP resulted in the incorporation of 32P into eight additional proteins besides CB9 that could be separated from the membranes by centrifugation in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We suggest that phosphorylation of CB9 or these additional eight proteins may regulate events underlying exocytosis in the chromaffin cell.     

Phosphorylation of the skeletal muscle AMP-deaminase by protein kinase C

Protein kinase C catalyzes phosphorylation of the rat skeletal muscle AMP-deaminase in the presence of calcium ions and phosphatidylserine. At the same time, the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate AMP-deaminase. Ca2+, phosphatidylserine-dependent phosphorylation decreases three-fold (from 0.6 to 0.2 mM) the Km value and does not affect Vmax. Protein kinase C-induced phosphorylation of AMP-deaminase, besides ADP-ribosylation, is suggested to be involved in regulating the AMP-deaminase activity in vivo.     

Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.     

Phosphorylation of aortic plasma membranes by protein kinase C

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca²⁺ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca^2– and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca²⁺.Abbreviations PKC Protein Kinase C - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - K_m Michaelis constant - NBT Nitro-Blue Tetrazolium - BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate     

Phosphorylation of bovine platelet myosin by protein kinase C

Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation.     

Phosphorylation of ras oncogene product by protein kinase C

The Harvey (H)-ras oncogene product, p21, can be phosphorylated by protein kinase C in vitro at sites distinct from the site of autophosphorylation of p21. Serine was found to be the main phosphate acceptor. Kinetic studies revealed a high apparent affinity but a much lower turnover for the phosphorylation of p21 as compared with that of the phosphorylation of histone by protein kinase C. Indirect association between protein kinase C and p21 was suggested by the co-immunoprecipitation of both proteins with either anti-protein kinase C or anti-p21 antibodies.     

Phosphorylation of protein kinase C by casein kinase-1

Because phosphorylation of protein kinase C (PKC) may provide a mechanism for regulation of this enzyme, we have examined the ability of two other kinases to phosphorylate PKC. Our results show that casein kinase 1 (CK-1), but not casein kinase 2 (CK-2), can phosphorylate PKC in the absence of Ca2+ and phospholipids. The 32P incorporation into PKC in the presence of Ca2+ and phospholipids is also enhanced by CK-1.     

Phosphorylation of bovine cardiac C-protein by protein kinase C

C-protein, a thick filament-associated protein, has been isolated from bovine myocardium and found to be a substrate in vitro of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C). Incorporation of approximately 1.6 mol Pi/mol C-protein was observed. This phosphorylation was dependent on both Ca2+ and a phospholipid (L-alpha-phosphatidyl-L-serine was used). Phosphate incorporation specifically into C-protein was verified by SDS-polyacrylamide gel electrophoresis and autoradiography and was almost exclusively into serine residues (86.9%), with only a small amount of phosphothreonine (13.1%) and no phosphotyrosine being detected. Two-dimensional thin-layer electrophoresis of a chymotryptic digest of phosphorylated C-protein indicated site specificity of phosphorylation. Cardiac C-protein is known to be a substrate of cAMP-dependent protein kinase both in vitro and in vivo (Jeacocke, S.A. and England, P.J. (1980) FEBS Lett. 122, 129-132). Isolated bovine cardiac C-protein was rapidly phosphorylated, to the extent of 5 mol/mol, by the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation catalyzed by these two protein kinases was not additive, suggesting that the sites phosphorylated by protein kinase C are also phosphorylated by cAMP-dependent protein kinase. Chicken cardiac muscle has also been shown to contain a Ca2+, calmodulin-dependent protein kinase which phosphorylates C-protein (Hartzell, H.C. and Glass, D.B. (1984) J. Biol. Chem. 259, 15587-15596). The physiological role of cardiac C-protein may therefore be subject to regulation by multiple protein kinases.     

Phosphorylation of rhodopsin by protein kinase C in vitro

Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium-stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings.