Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythorocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin_kinase according and site-specificity criteria. The soluble enzyme shows an M_r of about 30 000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32 000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that has been phosphorylated with [γ-³²P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21 500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with ³²P_i. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Phosphatidyl inositol inhibition of a sperm cyclic AMP-independent protein kinase

Phosphatidyl inositol has been found to inhibit strongly the activity of a cyclic AMP-independent protein kinase located on the external surface of goat epididymal intact spermatozoa. Phosphatidyl inositol at a concentration as low as 10 micrograms/ml inhibited nearly 50% of the ecto-kinase activity for the phosphorylation of the exogenous protein substrate: casein. Phosphatidyl ethanolamine at a relatively high concentration (125 micrograms/ml) inhibited slightly (approx 25%) the activity of the enzyme whereas other phospholipids: phosphatidyl serine and choline, diacyl glycerol, phosphatidic acid and myo-inositol-2-phosphate had no appreciable effect on the kinase activity. Phosphatidyl inositol has also served as a potent inhibitor of the phosphorylation of sperm ecto-phosphoproteins by the endogenous kinase activity of intact spermatozoa. By thin layer chromatography it has been shown that the observed inhibitory effect of the phospholipid was not due to any impurities or degraded products of phosphatidyl inositol. Phosphatidyl inositol inhibited the kinase activity noncompetitively with respect to casein and Mg2+ but uncompetitively with respect to ATP. The results raised the possibility that phosphatidyl inositol-mediated high affinity inhibition of protein kinase(s), may constitute a novel mechanism for the regulatory actins of the phospholipid in mammalian cells.     

A cyclic AMP-independent protein kinase from Candida albicans.

A cyclic AMP-independent protein kinase which phosphorylates casein was purified to homogeneity from Candida albicans by affinity and ion-exchange chromatography. This protein kinase exhibits maximal activity with casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The Mr of the purified enzyme is 115,000, as determined by h.p.l.c. It migrates as a single band on gel electrophoresis and has three non-identical subunits, of Mr 44,000, 28,500 and 26,000, as determined by SDS/polyacrylamide-gel electrophoresis. This enzyme is insensitive to heparin, but is inhibited by polyamines. Furthermore, it is sensitive to thermal denaturation and to thiol reagents.     

Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei

A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.     

Characterization and comparison of the soluble and membrane-bound cyclic AMP-dependent protein kinase from swine kidney

The catalytic subunits of adenosine 3′:5′ monophosphate-dependent protein kinase (ATP:protein transferase, E.C. 2.7.1.1.37) from the soluble and membrane fractions of swine kidney were purified to homogeneity by a new procedure and their structural, kinetic and immunological properties were compared. The specific activities of the purified enzymes were 2.35 and 2.6 µmol/min/mg of protein, with histone as the substrate. Both preparations contained a single polypeptide chain, and only one band was observed upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of both enzymes determined by gel electrophoresis was 42 000 ± 1000, and sedimentation equilibrium yielded a value of 41000 ± 800. Analysis by sedimentation velocity showed the presence of a single peak with and S_20,w of 3.1 ± 0.2 for each preparation. The amino acid compositions are very similar, and each enzyme contains about one residue of cysteine which is essential for enzymatic activity. ATP and Mg²⁺ protect both enzymes from inhibition by thiol specific reagents to the same extent. The catalytic subunits have similar apparent K m's for protein substrates. The enzymes exhibit single completely confluent precipitin lines when examined by immunodiffusion and the particulate catalytic subunit competitively displaces the soluble ¹²⁵I-catalytic subunit in homologous radioimmunoassays. The soluble and particulate ¹²⁵I-catalytic subunits bind to the regulatory subunits in the washed plasma membranes with attendent loss of kinase activity, which could be reversed by cyclic AMP. The results of experiments with kidney cortex slices treated with parathyroid hormone, epinephrine or dibutyryl cyclic AMP showed the translocation of phosphotransferase activity from the cytosol to the particulate membrane fraction. Taken collectively, these observations suggest that only one form of the catalytic subunit of cyclic AMP-dependent protein kinase is present in swine kidney, and that it may exchange between the cytosol and membrane fractions in response to specific physiological signals.     

Actin kinase: a protein kinase that phosphorylates actin of fragmin-actin complex.

Actin of fragmin-actin complex is phosphorylated by an endogenous kinase from plasmodium of Physarum polycephalum. The phosphorylation abolishes the nucleation and capping activities of fragmin-actin complex. The kinase has been purified and termed actin kinase [Furuhashi, K. & Hatano, S. (1990) J. Cell Biol. 111, 1081-1087]. Enzymatic properties of the purified actin kinase were studied in detail. Actin kinase exhibited the highest activity under conditions physiological for the plasmodium (30 mM KCl, 6 mM MgCl2, pH 7.0). The Vmax and the Km of the enzyme for ATP were about 83 mumol/min/mg and 25 microM, respectively. The Km for fragmin-actin complex was 190 nM. The purified actin kinase phosphorylated actin of fragmin-actin complex at a constant rate regardless of Ca2+ concentration. Similarly, 2 microM cAMP, 2 microM cGMP, 2 micrograms/ml calmodulin in the presence of Ca2+ or 1 mM GTP showed no effect on the activity of the purified enzyme. Actin kinase did not phosphorylate histone H1, H2B, alpha-casein, or beta-casein, suggesting that actin kinase is a new kind of protein kinase which specifically phosphorylates actin of the fragmin-actin complex.     

Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei.

A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.     

Evidence for ecto-protein kinase activity that phosphorylates Kemptide in a cyclic AMP-dependent mode

The heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) is a synthetic construct of a substrate for cAMP-dependent protein kinase (PK). In this work we show that Kemptide has all the properties of a cytophilic substrate, i.e. it is a molecule preserving cell membrane intactness when added to cultured cells. Kemptide thus satisfies the prerequisites for employment in assays for cell surface-located ecto-PK activity. Different types of intact cells catalyze the phosphorylation of Kemptide in the presence of extracellular ATP and cAMP with Km values of 3-4 microM for Kemptide. Kemptide phosphorylation was influenced by PKI, the inhibitory protein specific for cAMP-PK. The results of comparative experiments with intact cells and with cell extracts demonstrate the ectoenzyme nature of this cAMP-PK. Further, the possibility was ruled out of a transfer of enzyme activity from damaged cells to the surface of intact cells. The anchorage of the surface cAMP-PK activity to the plasma membrane appears to be relatively stable since (i) cell supernatants, obtained after preincubation of intact cells with cAMP or Kemptide, did not show Kemptide phosphorylation, and (ii) the cAMP-dependent PK activity remained with cells even after five consecutive washes with cAMP or Kemptide. This is in contrast to the ecto-cAMP-independent phosvitin/casein type PK (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025) which is released from intact cells through the addition of substrate. Data are presented which show that both ectokinase activities are exhibited independently. In conjunction with published evidence for an active export of cAMP from cells as well as for the appearance of extracellular ATP the demonstration of an ecto-cAMP-PK further supports the potential of PK for intercellular regulation. The potential of ecto-cAMP-PK is demonstrated by its ability to phosphorylate biologically active forms of atrial natriuretic peptide, the atrial natriuretic peptide, which possesses the specific sequence for a cAMP-PK-catalyzed phosphorylation.     

A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli

The membrane-bound diacylglycerol kinase from Swiss 3T3 cells (M-DG kinase) was characterized with a mixed micellar assay system, and compared with the cytosolic diacylglycerol kinase from 3T3 cells and with the membrane-bound diacylglycerol kinase from Escherichia coli. M-DG kinase selectively phosphorylated arachidonoyl-diacylglycerols, at a rate 2- to 8-fold higher than that for other naturally occurring long-chain diacylglycerols. In contrast, the cytosolic 3T3 enzyme exhibited little or no selectivity among long-chain diacylglycerols but had higher activity with more soluble substrates such as 1,2-didecanoylglycerol. Comparison of the properties of M-DG kinase with those of the bacterial membrane-bound enzyme revealed that selectivity for arachidonoyl-diacylglycerol was unique to the mammalian enzyme. All three kinases were activated by phosphatidylserine, but activation did not alter the arachidonoyl selectivity of M-DG kinase. Phosphatidylserine activated M-DG kinase by increasing Vm and decreasing the apparent Km for diacylglycerol. High concentrations of diacylglycerol reduced the Ka for phosphatidylserine, but did not abolish the phosphatidylserine requirement for maximum activity. Examination of the thermal lability of M-DG kinase revealed that this enzyme was rapidly and selectively inactivated by preincubation with its preferred substrate. This novel effect may have obscured previous attempts to discern substrate selectivity. Taken together, the results provide evidence that M-DG kinase is an arachidonoyl-diacylglycerol kinase that may participate in the formation of arachidonoyl-enriched species of phosphatidylinositol.     

Activation of a cyclic AMP-independent protein kinase by an endogenous ATP-requiring protease from lymphosarcoma cells

The activation of a cyclic AMP-independent protein kinase by an endogenous protease is described. The H4 phosphotransferase (Masaracchia, R. A., Kemp, B., and Walsh, D. A. (1977) J. Biol. Chem. 252, 7109-7117) from lymphosarcoma cells was isolated in a nonactive form. Activation required ATP and Mg2+ and was shown to be time-dependent. Although Mn2+ was capable of substituting for Mg2+ in the protein kinase reaction, no activation was observed when Mn2+ replaced Mg2+. The protein substrate histone H4 inhibited phosphotransferase activation at concentrations greater than 60 microM. The inhibition was complete in the presence of 100 microM H4. Comparable concentrations of bovine serum albumin did not inhibit the activation. The selective dependence on Mg2+ suggested separate activating and phosphotransferase activities. This was confirmed by heat denaturation in which the activation reaction was shown to be more sensitive to heat inactivation than was the phosphotransferase reaction. The activating enzyme was separated from the protein kinase by column chromatofocusing in the pH range 7-4. The pI of the activating enzyme was greater than 7.0. The pI values of the activated and nonactivated phosphotransferase were 4.8 and 5.3, respectively. The apparent molecular weight of the nonactivated phosphotransferase was 68,000; the activated enzyme was eluted from an S-200 Sephadex column with an apparent Mr = 52,000. Despite many similarities to a protease-activated Ca2+/phospholipid-dependent enzyme isolated from lymphocytes (Ogawa, Y., Takai, Y., Kawahara, Y., Kimura, S., and Nishizuka, Y. (1981) J. Immunol. 127, 1369-1374), the H4 phosphotransferase was not activated by Ca2+, phospholipids, or any combination thereof.     

Phosphorylation of ribosomal protein S6 at multiple sites by a cyclic AMP-independent protein kinase from lymphoid cells

Ribosomes prepared from murine lymphosarcoma cells were phosphorylated by a cyclic AMP-independent protein kinase designated H4P kinase. H4P kinase was isolated as an inactive enzyme which was activated by Mg2+-ATP and an endogenous converting enzyme. In the absence of preactivation by Mg2+-ATP and an endogenous converting enzyme, H4P kinase catalyzed phosphorylation of 80, 60, and 40 S ribosomal subunits at a low rate. After activation, the H4P kinase selectively catalyzed phosphorylation of the S 6 protein in the 40 S ribosomal subunit. Under the assay conditions selected, at least 90% of the [32P]phosphate transferred to the 40 S ribosomal preparation was incorporated into S 6. The apparent Km for 40 S subunits phosphorylated by H4P kinase was 7.2 microM. The calculated Vmax was 50 nmol of Pi transferred per min/mg. Exhaustive phosphorylation of 40 S subunits resulted in incorporation of 3 mol of phosphate/mol of S 6, in contrast to results reported previously which indicated 0.3 mol of phosphate was transferred by a similar enzyme from reticulocyte (Del Grande, R. W., and Traugh, J. A. (1982) Eur. J. Biochem. 123, 421-428). These data are consistent with a potential role for H4P kinase in the insulin-mediated phosphorylation of S 6 at multiple sites.     

Purification of a protein kinase from human Namalwa cells that phosphorylates topoisomerase I

A nuclear protein kinase which phosphorylates phosphoprotein $\text{110}\text{8.4}$, recently identified as topoisomerase I, has been purified approximately 330 fold from a 10 mM Tris extract of human Namalwa cells. The kinase wás chromatographed on DEAE-Sephacel and further purified by affinity chromatography on phosvitin-Sepharose. The protein kinase exhibited a high affinity (Km = 0.3 μM) for topoisomerase I; its affinity for phosvitin was approximately 100 fold lower (Km = 25 μM).     

Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY

Phosphorylation of circadian clock proteins represents a major regulatory step that controls circadian clocks. In Neurospora, the circadian clock protein FREQUENCY (FRQ) is progressively phosphorylated over time, and its level decreases when it is hyperphosphorylated. In this study, we showed that most of the kinase activity phosphorylating FRQ in vitro was calcium/calmodulin-dependent, and the endogenous FRQ in the Neurospora extracts was phosphorylated by a Ca/CaM-dependent kinase-like activity. From Neurospora cell extracts, an approximately 50-kDa Ca/CaM-dependent kinase (CAMK-1) that can specifically phosphorylate FRQ was purified. In vitro, this kinase accounts for near half of the FRQ kinase activity, and it can phosphorylate the FRQ region that contains the three known functionally important phosphorylation sites. To understand the function of camk-1 in vivo, it was disrupted in Neurospora by gene replacement. After germination from ascospores, the camk-1 null strains grew slowly, indicating that CAMK-1 plays an important role in growth and development of Neurospora. This phenotype was transient however, revealing redundancy in the system. Analysis of the camk-1 null strain revealed that the deletion of camk-1 affected phase, period, and light-induced phase shifting of the circadian conidiation rhythm. Taken together, our results suggest that multiple kinases may phosphorylate FRQ in vivo.     

Identification of a protein kinase activity in rabbit reticulocytes that phosphorylates the mRNA cap binding protein

The 25 kDa mRNA cap binding protein can be purified in a partially phosphorylated state and the extent of its phosphorylation appears to be regulated during heat shock and mitosis in mammalian cells. We demonstrated that a nonabundant serine protein kinase activity exists in rabbit reticulocytes that phosphorylates the 25 kDa cap binding protein in both the free (eIF-4E) and complexed (eIF-4F) state. This kinase was not inhibited by the cAMP-dependent protein kinase inhibitory peptide IAAGRTGRRNAIHDILVAA, did not phosphorylate S6 ribosomal protein, did not phosphorylate p220 of eIF-4F as protein kinase C does and no other substrates for this kinase were apparent in reticulocyte ribosomal salt wash. The molecular identity of this kinase, the specific site(s) of eIF-4E that it phosphorylates and its in vivo regulatory role remain to be studied.     

Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates

Summary The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examining competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s).The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP.Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.     

Goodpasture antigen-binding protein is a soluble exportable protein that interacts with type IV collagen. Identification of novel membrane-bound isoforms

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at least two GPBP isoforms resulting from canonical (77-kDa) and noncanonical (91-kDa) mRNA translation initiation. The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. These and previous data implicate GPBPs in a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane.     

Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein

Escherichia coli NCR91 synthesizes a mutant form of catabolite gene activator protein (CAP) in which alanine 144 is replaced by threonine. This mutant, which also lacks adenylate cyclase activity, has a CAP phenotype; in the absence of cAMP it is able to express genes that normally require cAMP. CAP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild type CAP X cAMP complex. X-ray diffraction data were measured to 2.4-A resolution and the CAP91 structure was determined using initial model phases from the wild type structure. A difference Fourier map calculated between CAP91 and wild type showed the 2 alanine to threonine sequence changes in the dimer and also a change in orientation of cysteine 178 in one of the subunits. The CAP91 coordinates were refined by restrained least squares to an R factor of 0.186. Differences in the atomic positions of the wild type and mutant protein structures were analyzed by a vector averaging technique. There were small changes that included concerted motions in the small domains, in the hinge between the two domains and in an adjacent loop between beta-strands 4 and 5. The mutation at residue 144 apparently causes changes in the position of some protein atoms that are distal to the mutation site.