Neuronal Cdc2-like protein kinase (Cdk5/p25) is associated with protein phosphatase 1 and phosphorylates inhibitor-2.

Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1.I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr(72) by glycogen synthase kinase 3 (GSK3). We have found that PP1.I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only approximately 29% of PP1 activity and indicated that GSK3 is not the sole PP1.I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1.I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a approximately 450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr(72) and activated PP1.I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28-41. Our data demonstrate that in brain extract PP1.I-2 and NCLK are associated within a complex of approximately 450 kDa and suggest that NCLK is one of the PP1.I-2-activating kinases in the mammalian brain.

14-3-3zeta is an effector of tau protein phosphorylation.

Neurofibrillary tangles associated with Alzheimer's disease are composed mainly of paired helical filaments that are formed by the aggregation of abnormally phosphorylated microtubule-associated protein tau. 14-3-3, a highly conserved protein family that exists as seven isoforms and regulates diverse cellular processes is present in neurofibrillary tangles (Layfield, R., Fergusson, J., Aitken, A., Lowe, J., Landon, M., Mayer, R. J. (1996) Neurosci. Lett. 209, 57-60). The role of 14-3-3 in Alzheimer's disease pathogenesis is not known. In this study, we found that the 14-3-3zeta isoform is associated with tau in brain extract and profoundly stimulates cAMP-dependent protein kinase catalyzed in vitro phosphorylation on Ser(262)/Ser(356) located within the microtubule-binding region of tau. 14-3-3zeta binds to both phosphorylated and nonphosphorylated tau, and the binding site is located within the microtubule-binding region of tau. From brain extract, 14-3-3zeta co-purifies with microtubules, and tubulin blocks 14-3-3zeta-tau binding. Among four 14-3-3 isoforms tested, beta and zeta but not gamma and epsilon associate with tau. Our data suggest that 14-3-3zeta is a tau protein effector and may be involved in the abnormal tau phosphorylation occurring during Alzheimer's disease ontogeny.

Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor.

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase that phosphorylates Ser(67) had not been identified and the physiological role of Ser(67) phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione S-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values approximately 9.5 and 13. 8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.

Interaction of neuronal Cdc2-like protein kinase with microtubule-associated protein tau.

Neuronal Cdc2-like protein kinase (NCLK), a approximately 58-kDa heterodimer, was isolated from neuronal microtubules (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901). The biochemical nature of NCLK-microtubule association is not known. In this study we found that NCLK is released from microtubules upon microtubule disassembly as a 450-kDa species. The 450-kDa species is an NCLK.tau complex, and NCLK-bound tau is in a nonphosphorylated state. Tau phosphorylation causes NCLK.tau complex dissociation, and phosphorylated tau does not bind to NCLK. In vitro, the Cdk5 subunit of NCLK binds to the microtubule-binding region of tau and NCLK associates with microtubules only in the presence of tau. Our data indicate that in brain extract NCLK is complexed with tau in a tau phosphorylation-dependent manner and that tau anchors NCLK to microtubules. Recently NCLK has been suggested to be aberrantly activated and to hyperphosphorylate tau in Alzheimer's disease brain (Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P, and Tsai, L.-H. (1999) Nature 402, 615-622). Our findings may explain why in Alzheimer's disease NCLK specifically hyperphosphorylates tau, although this kinase has a number of protein substrates in the brain.

The topography and subcellular distribution of mitogen-activated protein kinase kinase1 (MEK1) in adult rat brain and differentiating PC12 cells.

Mitogen-activated protein kinase signal transduction pathway involved in the regulation of proliferation and differentiation of various mammalian cells consists of a sequential activation of three protein kinases, Raf, mitogen-activated protein kinase kinase, and mitogen-activated protein kinase. These kinases are highly expressed in brain and play an important role in neuronal signalling. In this study, to further characterize mitogen-activated protein kinase signalling pathway in brain, we have elucidated the topography and subcellular distribution of mitogen-activated protein kinase kinasel in adult rat brain and differentiating PC12 cells. Our immunohistochemical data indicate that mitogen-activated protein kinase kinase1 is widely distributed throughout the brain and expressed prominently in cortex, hippocampus, brainstem, hypothalamus and cerebellum. In these areas of brain mitogen-activated protein kinase kinasel is exclusively neuronal in origin and is localized within perikarya and dendrites. Confocal microscopy data has determined that a portion of mitogen-activated protein kinase kinase1 in rat brain is co-localized with microtubules. This co-localization was observed only within neuritic shaft and cilia of ventricular ependymal cells. In nerve growth factor-induced differentiating PC12 cells, mitogen-activated protein kinase kinase1 displays co-localization with microtubules within proximal regions of neuritic shafts and their junctions with the cell somas. From bovine brain extract, mitogen-activated protein kinase kinasel co-purifies with microtubules. In vitro kinase assay detected mitogen-activated protein kinase kinase1 activity within purified microtubules. These observations indicate that mitogen-activated protein kinase kinase1 is associated with microtubules within some specialized compartments of the brain and microtubule-associated mitogen-activated protein kinase kinase1 is catalytically active.

Heparin-induced conformational change in microtubule-associated protein Tau as detected by chemical cross-linking and phosphopeptide mapping.

In Alzheimer's disease, microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments. Sulfated glycosaminoglycans such as heparin and heparan sulfate were shown to accumulate in pretangle neurons, stimulate in vitro tau phosphorylation, and cause tau aggregation into paired helical filament-like filaments. The sulfated glycosaminoglycan-tau interaction was suggested to be the central event in the development of neuropathology in Alzheimer's disease brain (Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996) Nature 383, 550-553). The biochemical mechanism by which sulfated glycosaminoglycans stimulate tau phosphorylation and cause tau aggregation remains unclear. In this study, disuccinimidyl suberate (DSS), a bifunctional chemical cross-linker, cross-linked tau dimers, tetramers, high molecular size aggregates, and two tau species of sizes 72 and 83 kDa in the presence of heparin. In the absence of heparin only dimeric tau was cross-linked by DSS. Fast protein liquid chromatography gel filtration revealed that 72- and 83-kDa species were formed by intramolecular cross-linking of tau by DSS. These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species. Heparin-induced structural changes in tau molecule depended on time of heparin exposure. Dimerization and tetramerization peaked at 48 h, whereas conformational change was completed within 30 min of heparin exposure. Heparin exposure beyond 48 h caused an abrupt aggregation of tau into high molecular size species. Heparin stimulated tau phosphorylation by neuronal cdc2-like kinase (NCLK) and cAMP-dependent protein kinase. Phosphopeptide mapping and phosphopeptide sequencing revealed that tau is phosphorylated by NCLK on Thr212 and Thr231 and by cAMP-dependent protein kinase on Ser262 only in the presence of heparin. Heparin stimulation of tau phosphorylation by NCLK showed dependence on time of heparin exposure and correlated with the heparin-induced conformational change of tau. Our data suggest that heparin-induced conformational change exposes new sites for phosphorylation within tau molecule.

Phosphorylation sensitizes microtubule-associated protein tau to Al(3+)-induced aggregation.

In Alzheimer's disease the microtubule-associated protein tau becomes hyperphosphorylated and aggregates into paired helical filaments (PHFs). Although the biochemical basis of the aggregation of tau into PHFs is not very clear, Al3+ has been suggested to play some role. Previous studies have shown that Al3+ alters the phosphorylation state and causes aggregation of tau in experimental animals and cultured neurons. In this study Al3+ inhibited phosphorylation of tau by neuronal cdc2-like kinase and dephosphorylation of phosphorylated tau by phosphatase 2B. These inhibitions are very likely due to Al(3+)-induced aggregations of various proteins present in phosphorylation/dephosphorylation assay mixtures since Al3+ caused aggregations of all proteins examined. Furthermore, compared to other proteins, tau displayed only an average sensitivity towards Al(3+)-induced aggregation. However upon phosphorylation, tau's sensitivity towards Al3+ increased 3.5 fold. In the presence of the metal chelator EDTA, Al(3+)-induced aggregates of tau became soluble, whereas Al(3+)-induced phosphorylated tau aggregates were insoluble in the buffer containing EDTA and remained insensitive to proteolysis. Our data suggest that phosphorylation sensitizes tau to Al3+ and phosphorylated tau transforms irreversibly into a phosphatase and protease resistant aggregate in presence of this metal ion.

Phosphorylation by neuronal cdc2-like protein kinase promotes dimerization of Tau protein in vitro.

In Alzheimer's disease, the microtubule-associated protein tau forms paired helical filaments (PHFs) that are the major structural component of neurofibrillary tangles. Although tau isolated from PHFs (PHF-tau) is abnormally phosphorylated, the role of this abnormal phosphorylation in PHF assembly is not known. Previously, neuronal cdc2-like protein kinase (NCLK) was shown to phosphorylate tau on sites that are abnormally phosphorylated in PHF-tau (Paudel, H. K., Lew, J., Ali, Z., and Wang, J. H. (1993) J. Biol. Chem. 268, 23512-23518). In this study, phosphorylation by NCLK was found to promote dimerization of recombinant human tau (R-tau) and brain tau (B-tau) purified from brain extract. Chemical cross-linking by disuccinimidyl suberate (DSS), a homobifunctional chemical cross-linker that specifically cross-linked R-tau dimers, and a Superose 12 gel filtration chromatography revealed that R-tau preparations contain mixtures of monomeric and dimeric R-tau species. When the structure of NCLK-phosphorylated R-tau was studied by a similar approach, DSS preferentially cross-linked the phosphorylated R-tau over the nonphosphorylated R-tau, and the phosphorylated R-tau eluted as a dimeric species from the gel filtration column. Phosphorylated R-tau became resistant to DSS upon dephosphorylation and was recovered as a monomeric species from the gel filtration column. In the presence of a low concentration of dithiothreitol (1.65 microM), R-tau formed disulfide cross-linked R-tau dimers. When compared, phosphorylated R-tau formed more disulfide cross-linked dimers than the nonphosphorylated R-tau. B-tau also was specifically cross-linked to dimers by DSS. When B-tau and NCLK-phosphorylated B-tau were treated with DSS, phosphorylated B-tau was preferentially cross-linked over nonphosphorylated counterpart. Taken together, these results suggest that phosphorylation by NCLK promotes dimerization and formation of disulfide cross-linked tau dimers, which is suggested to be the key step leading to PHF assembly (Schweers, O., Mandelkow, E.-M., Biernat, J., and Mandelkow, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8463-8467).

The regulatory Ser262 of microtubule-associated protein tau is phosphorylated by phosphorylase kinase.

Abnormally phosphorylated tau is the major component of paired helical filaments found in the brains of patients suffering from Alzheimer's disease. Therefore, the identification of kinases that phosphorylate tau is of considerable interest. A DEAE-Sepharose column resolved porcine brain extract into five tau kinase activity peaks. Among these peaks, two were completely inhibited by EGTA, indicating that these two activity peaks contained Ca2+-dependent tau kinases. One of the above two Ca2+-dependent tau kinase activity peaks also contained phosphorylase kinase activity. The tau kinase and phosphorylase kinase activities associated with this peak could not be separated from each other by Superose 12 gel filtration, hydroxylapatite, and calmodulin-agarose affinity chromatographies. Phosphorylase kinase, purified from rabbit skeletal muscle, phosphorylated tau to a stoichiometry of 2.1 mol of phosphate/mol of tau and converted tau to a species with a retarded mobility on SDS-polyacrylamide gel electrophoresis. The apparent Km and kcat values for tau phosphorylation by muscle phosphorylase kinase were 6.9 microM and 47.4 min-1, respectively. As a substrate of muscle phosphorylase kinase, phosphorylase was eight times better than tau. Sequence analyses of tryptic and thermolytic phosphopeptides derived from tau phosphorylated by muscle phosphorylase kinase revealed five phosphorylation sites, Ser237, Ser262, Ser285, Ser305, and Ser352. Among these sites, Ser262 was previously shown to be phosphorylated in human tau from fetal, adult, and Alzheimer's diseased brains (Seubert, P., Mawal-Dewan, M., Barbour, R., Jakes, R., Goedert, M., Johnson, G. V. W., Litersky, J. M., Schenk, D., Lieberburg, I., Trojanowski, J. Q., and Lee, V. M. Y. (1995) J. Biol. Chem. 270, 18917-18922); and its phosphorylation abolished tau's binding to microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Slot-blot analysis using a monoclonal antibody against muscle phosphorylase kinase and an activity assay using phosphorylase revealed that phosphorylase kinase was present in microtubules extensively purified by repeated cycles of polymerization and depolymerization. Taken together, these results suggest that in neurons, phosphorylase kinase may be one of the kinases that participate in the phosphorylation of tau.

Brain proline-directed protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments.

Brain proline-directed protein kinase (BPDK), which contains a catalytic subunit homologous to and displaying site-specific phosphorylation similar to p34cdc2 kinase (Lew, J., Winkfein, R. J., Paudel, H. K., and Wang, J. H. (1992) J. Biol. Chem. 267, 25922-25926), has been examined for possible involvement in tau phosphorylation. Immunoblot analyses using peptide antibodies specific for BPDK have revealed the presence of the kinase in bovine brain microtubules purified extensively by repeated polymerization and depolymerization cycles. When the microtubule proteins are separated into the tubulin and microtubule-associated protein fractions, BPDK is found exclusively in the latter fraction. BPDK phosphorylates both tau and MAP2, the former protein being phosphorylated to a stoichiometry of 3.8 mol of phosphate/mol of tau. Analysis of the phosphopeptides isolated from the tryptic digest of the phosphorylated bovine tau has revealed seven phosphorylation sites. Based on the sequence alignment between bovine and human tau proteins, these sites correspond to Ser-195, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, and Ser-404 of human tau. Mass spectrometric analysis of the tau protein isolated from Alzheimer's paired helical filaments (PHFs) has determined three abnormal phosphorylation sites and two phosphopeptides containing a total of five abnormal phosphates (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 267, 17047-17054). Two of the sites in tau phosphorylated by BPDK, Thr-231 and Ser-235, are among the abnormal phosphorylation sites, and the other sites phosphorylated by BPDK are within phosphopeptides from PHF-tau. These results suggest that BPDK may be one of the kinases responsible for the abnormal phosphorylation-associated PHF-tau.

The model calmodulin-binding peptide melittin inhibits phosphorylase kinase by interacting with its catalytic center.

The inhibition by melittin, a model calmodulin-binding peptide, of phosphorylase kinase, which contains an intrinsic calmodulin subunit, has been characterized in detail. The inhibition was competitive with respect to phosphorylase b for both the phosphorylase kinase holoenzyme and its isolated catalytic gamma-subunit (minus calmodulin), and the ratios of the Km for phosphorylase to the Ki for melittin were similar for both forms of the kinase. These findings indicate that inhibition of the phosphorylase kinase holoenzyme by melittin is caused predominantly by its interaction with the catalytic subunit of the enzyme, and not with the endogenous calmodulin subunit. Further proof that melittin interacts directly with the catalytic site was obtained when it was observed that melittin was also a substrate for phosphorylase kinase, with a Km that was less than that for phosphorylase b, although the kcat/Km specificity constant was only 1/200th of that for phosphorylase. The apparent tight binding of melittin to the kinase active site could not be readily rationalized by conventional comparison of sequence similarity between melittin and phosphorylase; however, considerable sequence similarity, centered around the convertible seryl residue of phosphorylase, was observed when the sequences were aligned in reversed polarity. The possible regulatory significance of the direct interaction of the catalytic site of this Ca(2+)-dependent kinase with a calmodulin-binding peptide is discussed.

Phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of neuronal tissue-specific proteins B-50 (GAP-43) and neurogranin.

Neuronal tissue-specific proteins B-50 (GAP-43, neuromodulin) and neurogranin are phosphorylated by phosphorylase kinase with stoichiometries of 0.4 and 0.5 mol of phosphate/mol of protein, respectively. The apparent Km and kcat values determined at pH 8.2 for neurogranin phosphorylation are 28.4 microM and 139.3 min-1, respectively, and for B-50 phosphorylation are 22.8 microM and 33.2 min-1, respectively. As a substrate of phosphorylase kinase, phosphorylase is approximately 44 and approximately 13 times better than B-50 and neurogranin, respectively. Both proteins are better substrates of protein kinase C than of phosphorylase kinase and are phosphorylated on a single site by phosphorylase kinase. The sequence analyses of tryptic phosphopeptides isolated from neurogranin and B-50 phosphorylated by phosphorylase kinase revealed the same amino acid sequence, IQASF, indicating that phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of B-50 and neurogranin previously known to be phosphorylated by protein kinase C (Coggins, P. J., and Zwiers, H. (1989) J. Neurochem. 53, 1895-1901; Baudier, J., Deloulme, J. C., Dorsselaer, A. V., Black, D., and Matthes, W. D. (1991) J. Biol. Chem. 266, 229-237). In rat brain synaptosomes, a relatively high phosphorylase kinase specific activity is detected, and approximately 32% activity is associated with synaptic membranes where B-50 is localized. In rat brain homogenate and synaptosomal membranes, phosphorylation of a protein that co-migrates with B-50 on SDS-polyacrylamide gel electrophoresis is enhanced in the presence of exogenous phosphorylase kinase.

Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2.

The carboxyl-terminal regions of neurofilament high (NF-H) and middle (NF-M) molecular weight proteins have been suggested to be phosphorylated in vivo by a p34cdc2-like protein kinase, on the basis of the in vivo phosphorylation site motif and in vitro phosphorylation of the proteins by p34cdc2 kinase (Hisanaga, S.I., Kusubata, M., Okumura, E. and Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803). A novel proline-directed protein kinase previously identified and purified from bovine brain has been found in this study to phosphorylate NF-H and NF-M at sites identical to those phosphorylated by HeLa cell p34cdc2 kinase. The proline-directed kinase is composed of a 33-kDa and a 25-kDa subunit. The 33-kDa kinase subunit was partially sequenced, and degenerate oligonucleotide primers corresponding to the amino acid sequence information were used to clone the subunit by polymerase chain reaction (PCR). Two overlapping PCR products comprised a complete open reading frame of 292 amino acids. The sequence contains all features of a protein kinase, suggesting that the 33-kDa peptide represents the catalytic subunit of the kinase. The 33-kDa subunit shows high and approximately equal homology to human p34cdc2 and human cdk2, with about 58 and 59% amino acid identity, respectively. These results suggest that the brain kinase represents a new category of the cdc2 family, and that some members of the cdc2 kinase family may have major functions unrelated to cell cycle control.

The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity.

Phosphorylase kinase from rabbit skeletal muscle has been found to have an intrinsic ATPase activity that occurs at a rate approximately 0.2% of that of its phosphorylase conversion activity and about three times that of its autophosphorylation activity. The characteristics of this ATPase activity were in all aspects tested essentially the same as the kinase's phosphorylase conversion activity. The ATPase requires Mg2+ and is dramatically stimulated by Ca2+ ions. At neutral pH there is a pronounced lag in the rate of product formation that is not present at alkaline pH, a condition that greatly stimulates both the phosphorylase conversion and ATPase activities. ATP is preferentially hydrolyzed over GTP and the Km for MgATP determined in the ATPase assay is 0.14 mM. ADP, an allosteric activator of phosphorylase conversion, also stimulates the ATPase activity, whereas beta-glycerophosphate, an inhibitor of phosphorylase conversion, is an inhibitor of the ATPase activity. Phosphorylation or partial proteolysis of the kinase, which are known to activate phosphorylase conversion, also activate the ATPase activity. Because the phosphorylase conversion and ATPase activities are regulated in parallel, we conclude that activation of the two catalytic activities must share a common underlying basis, namely an enhanced phosphotransferase activity that is independent of the phosphoryl acceptor.

Involvement of a histidine residue in the interaction between membrane-anchoring protein (QPs) and succinate dehydrogenase in mitochondrial succinate-ubiquinone reductase.

The involvement of a histidine residue of the membrane-anchoring protein (QPs) fraction in reconstitution of succinate dehydrogenase to form succinate-ubiquinone reductase is studied by using a histidine-modifying reagent, diethylpyrocarbonate (DEPC). A maximum inactivation of 80% of reconstitutive activity is obtained when QPs is treated with 1 mM DEPC at 0 degrees C for 30 min in 50 mM Tris-HCl (pH 7.0). DEPC also inactivates about 85% of intact succinate-ubiquinone reductase. The inactivation of succinate-ubiquinone reductase by DEPC is a result of the modification of essential histidine residues of succinate dehydrogenase. The inactivation is not a result of the modification of the histidine residue in QPs which is essential for interaction with succinate dehydrogenase because the QPs dissociated from the inactivated succinate-ubiquinone reductase is active in reconstitution with active succinate-dehydrogenase. Apparently, the essential histidine in QPs is shielded by succinate dehydrogenase and thus inaccessible to DEPC modification in succinate-ubiquinone reductase. The involvement of a histidine residue of QPs in interaction with succinate dehydrogenase is further evident by the presence of 553 nm shoulder on the alpha-absorption peak of reduced cytochrome b-560 (a characteristic of physical association of QPs with succinate dehydrogenase) in the DEPC-inactivated succinate-ubiquinone reductase. This shoulder disappears from a mixture of succinate dehydrogenase and DEPC-treated QPs when reduced with dithionite. About one histidine residue per molecule of QPs is modified in the DEPC-treated sample, suggesting that only one histidine residue is essential for interaction with succinate dehydrogenase. This essential histidine group is located in the smaller subunit (Mr 13,000) of QPs.

Functional and structural similarities between the inhibitory region of troponin I coded by exon VII and the calmodulin-binding regulatory region of the catalytic subunit of phosphorylase kinase.

A sequence homology has been noted between the carboxyl quarter of the catalytic gamma subunit of phosphorylase kinase and the region of troponin I coded by exon VII. Because this portion of troponin I contains the inhibitory region that interacts with actin and troponin C, we have examined whether the gamma subunit of phosphorylase kinase can functionally mimic troponin I by also interacting with actin and troponin C. We have found that troponin C not only activates the isolated gamma subunit of phosphorylase kinase but also binds with approximately the same affinity as calmodulin. Although actin had no effect on the activity of the gamma subunit alone, it did inhibit the activity of gamma-calmodulin and gamma-troponin C complexes. Conversely, the gamma subunit was able to inhibit actomyosin ATPase in a process that could be overcome by calmodulin. These results suggest that actin and calmodulin (or troponin C) compete for binding to the gamma subunit. Moreover, the structural and functional similarities between the gamma subunit and troponin I suggest that the gamma subunit of phosphorylase kinase may have evolved from the fusion of a protein kinase protogene with a progenitor of exon VII of troponin I.

The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin.

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, beta- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, beta- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only beta-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.

Renaturation of phosphorylase kinase activity from sodium dodecyl sulfate-polyacrylamide gels.

Phosphorylase kinase activity is renatured and detected in situ following electrophoresis of the denatured holoenzyme in a sodium dodecyl sulfate-polyacrylamide gel containing phosphorylase b that has been included in the gel polymerization according to the method of R. L. Geahlen et al. [(1986) Anal. Biochem. 153, 151-158]. Among the enzyme's four subunits, only gamma is catalytically active. When extract of rabbit muscle is electrophoresed and renatured in a similar manner, the phosphorylase-conversion activity is also associated only with a protein band that comigrates with the gamma subunit of phosphorylase kinase. This suggests that the gamma subunit of phosphorylase kinase may be the sole activity in rabbit muscle responsible for the phosphorylation of phosphorylase b. In an alternative method for the renaturation of activity from conventional sodium dodecyl sulfate-polyacrylamide gels, the subunits of the enzyme are visualized using 2.5 M KCl, excised from the gel, and eluted by diffusion into buffer containing sodium dodecyl sulfate, which is subsequently removed by acetone precipitation of the eluted subunits. Catalytic activity is recovered when the acetone precipitate of the extracted gamma subunit is dissolved in 6 M guanidine hydrochloride and diluted 50-fold into an activity assay. Inclusion of eluted alpha and beta subunits in the assay inhibits the activity of the gamma subunit, which supports our previous finding that the alpha and/or beta subunits suppress the activity of the catalytic gamma subunit [H. K. Paudel and G. M. Carlson (1987) J. Biol. Chem. 262, 11912-11915].

Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits.

The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma.

Purification, characterization, and the complete amino acid sequence of porcine pancreatic deoxyribonuclease.

Porcine pancreatic DNase has been purified to homogeneity. The polypeptide exhibits a single band of Mr = 34,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a glycoprotein containing glucosamine. The results of end group analyses show leucine at the NH2 terminus and alanine at the COOH terminus. The enzymatic properties of the purified porcine DNase are very similar to those of bovine and ovine DNases. The sequence data on the tryptic and chymotryptic peptides derived from CNBr fragments of porcine DNase, along with the results of automated Edman degradation of the intact polypeptide and of the two largest CNBr fragments, indicate the complete amino acid sequence of porcine DNase to be as follows:L-R- I-A-F-N-I-R-T-F-G-E-T-K-M-S-N-A-T-S-N-Y-I-V-R-I-L-S-R-Y-D-I-A-L-I-Q- E-V-R-D-S-H-L-T-A-V-G-K-L-L-N-E-L-N-Q-D-D-P-N-N-Y-H-H-V-V-S-E-P-L-G-R- S-T-Y-K-E-R-Y-L-F-V-F-R-P-N-Q-V-S-V-L-D-S-Y-L-Y-D-D-G-C-E-P-C-G-N-D-T- F-N-R-E-P-S-V-V-K-F-S-S-P-F-T-Q-V-K-E-F-A-I-V-P-L-H-A-A-P-S-D-A-A-A-E- I-N-S-L-Y-D-V-Y-L-N-V-R-Q-K-W-D-L-Q-D-I-M-L-M-G-D-F-N-A-G-C-S-Y-V-T- T-S-H-W-S-S-I-R-L-R-E-S-P-P-F-Q-W-L-I-P-D-T-A-D-T-T-V-S-S-H-T-C-A-Y- D-R-I-V-V-A-G-P-L-L-Q-R-A-V-V-P-D-S-A-A-P-F-D-F-Q-A-A-F-G-L-S-Q-E-T- A-L-A-I-S-D-H-Y-P-V-E-V-T-L-K-R-A. The polypeptide consists of 262 amino acid residues. One of the two disulfide loops links Cys-101 and Cys-104 and the other Cys-173 and Cys-209. Two carbohydrate side chains are attached at Asn-18 and Asn-106.