scholarly journals Activation by phosphorylation of phosphofructokinase from the annelid Lumbricus terrestris and comparison of phosphorylated sites in invertebrate phosphofructokinases

1996 ◽  
Vol 317 (2) ◽  
pp. 377-383 ◽  
Author(s):  
Ralph SCHALOSKE ◽  
Michael BIETHINGER ◽  
Linda A. FOTHERGILL-GILMORE ◽  
Hans Werner HOFER
Keyword(s):  
Protein Phosphatase ◽  
Helix Pomatia ◽  
Lumbricus Terrestris ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
N Terminus ◽  
Phosphatase 2A ◽  
Dependent Protein ◽  
Earthworm Lumbricus

Purified phosphofructokinase from the earthworm Lumbricus terrestris was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase from the same organism to an extent of approx. 0.5 mol/mol of subunit. Activation of the enzyme occurred in parallel to the incorporation of covalently bound phosphate and was reversed by the action of the catalytic subunit of protein phosphatase 2A. Phosphorylation decreased the co-operativity of fructose 6-phosphate saturation in the presence of inhibitory concentrations of ATP, and increased the apparent Vmax obtained with saturating concentrations of the activators 5´-AMP and fructose 2,6-bisphosphate. The phosphorylated sites of phosphofructokinase from L. terrestris and from two molluscs (Helix pomatia and Mytilus edulis) were sequenced and shown to exhibit distinct similarity to sequences located near to the N-terminus of nematode phosphofructokinases [Klein, Olson, Favreau, Wintertowed, Hatzenbuhler, Shea, Nulf and Geary (1991) Mol. Biochem. Parasitol. 48, 17–26].

scholarly journals Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis

2015 ◽  
Vol 35 (15) ◽  
pp. 2699-2713 ◽  
Author(s):  
Pauline Douglas ◽  
Ruiqiong Ye ◽  
Nicholas Morrice ◽  
Sébastien Britton ◽  
Laura Trinkle-Mulcahy ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Dependent Protein Kinase ◽  
Hnrnp U ◽  
Multiple Characteristics ◽  
Phosphatase 2A ◽  
Dependent Protein ◽  
Serine 59 ◽  
Attachment Factor

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis.

scholarly journals Fructose-2,6-bisphosphatase and 6-phosphofructo-2-kinase are separable in yeast

1987 ◽  
Vol 246 (3) ◽  
pp. 755-759 ◽  
Author(s):  
M Kretschmer ◽  
W Schellenberger ◽  
A Otto ◽  
R Kessler ◽  
E Hofmann
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Dependent Protein Kinase ◽  
Km Value ◽  
Phosphatase 2A ◽  
Dependent Protein

Fructose-2,6-bisphosphatase was purified from yeast and separated from 6-phosphofructo-2-kinase and alkaline phosphatase. The enzyme released Pi from the 2-position of fructose 2,6-bisphosphate and formed fructose 6-phosphate in stoichiometric amounts. The enzyme displays hyperbolic kinetics towards fructose 2,6-bisphosphate, with a Km value of 0.3 microM. It is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by L-glycerol 3-phosphate. Phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase causes inactivation, which is reversible by the action of protein phosphatase 2A.

Regulation of an epithelial chloride channel by direct phosphorylation and dephosphorylation

1992 ◽  
Vol 263 (1) ◽  
pp. C172-C175 ◽  
Author(s):  
A. L. Finn ◽  
M. L. Gaido ◽  
M. Dillard ◽  
D. L. Brautigan
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Lipid Bilayers ◽  
Okadaic Acid ◽  
Specific Antibody ◽  
Chloride Channel ◽  
Protein Phosphatase ◽  
Dependent Protein Kinase ◽  
Phosphatase 2A ◽  
Dependent Protein

A native chloride channel in Necturus gallbladder epithelial cells is opened by a theophylline-induced rise in cellular cyclic AMP and is closed by removal of theophylline or by addition of specific antibody; however, it does not close if okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is added. The purified channel reconstituted into lipid bilayers closes upon the addition of protein phosphatase 2A and is reopened by the addition of Mg-ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. These results indicate that the channel protein is purified in a phosphorylated state and that its functional characteristics are at least partly controlled by direct phosphorylation and dephosphorylation.

Protein phosphorylation and the control of glycogen metabolism in skeletal muscle

1983 ◽  
Vol 302 (1108) ◽  
pp. 13-25 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Cyclic Amp ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Catalytic Subunit ◽  
Glycogen Synthase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Glycogen metabolism in mammalian skeletal muscle is controlled by a regulatory network in which six protein kinases, four protein phosphatases and several thermostable regulatory proteins determine the activation state of glycogen phosphorylase and glycogen synthase, the rate-limiting enzymes of this process. Thirteen phosphorylation sites are involved, twelve of which have been isolated and sequenced and shown to be phosphorylated in vivo . The effects of adrenalin and insulin on the state of phosphorylation of each site have been determined. The neural control of glycogen metabolism is mediated by calcium ions and involves phosphorylase kinase, and a specific calmodulin-dependent glycogen synthase kinase. The β-adrenergic control of the system is mediated by cyclic AMP, and involves the phosphorylation of phosphorylase kinase, glycogen synthase and inhibitor 1 by cyclic-AMP-dependent protein kinase. Inhibitor 1 is a specific inhibitor of protein phosphatase 1, the major phosphatase involved in the control of glycogen metabolism. The stimulation of glycogen synthesis by insulin results from the dephosphorylation of glycogen synthase at sites (3 a + 3 b + 3 c ), which are introduced by the enzyme glycogen synthase kinase 3. The structure, regulation and substrate specificities of the protein phosphatases involved in glycogen metabolism are reviewed. Protein phosphatase 1 can exist in an inactive form termed the Mg-ATP-dependent protein phosphatase, which consists of a complex between the catalytic subunit and a thermostable protein termed inhibitor 2. Activation of this complex is catalysed by glycogen synthase kinase 3. It involves the phosphorylation of inhibitor 2 and its dissociation from the catalytic subunit. Protein phosphatase 2A can be resolved into three forms by ion exchange chromatography. These species contain the same catalytic subunit and other subunits that may have a regulatory function. Protein phosphatase 2B is a Ca 2+ -dependent enzyme composed of two subunits, A and B. Its activity is increased tenfold by calmodulin, which interacts with the A-subunit. The B-subunit is a Ca 2+ -binding protein that is homologous with calmodulin. Its N-terminus contains the unusual myristyl blocking group, only found previously in the catalytic subunit of cyclic-AMP-dependent protein kinase. Protein phosphatase 2C is a Mg 2+ -dependent enzyme that accounts for a very small proportion of the glycogen synthase phosphatase activity in skeletal muscle. It is likely to be involved in the regulation of other metabolic processes in vivo such as cholesterol synthesis. Recent evidence suggests that many of the proteins involved in the control of glycogen metabolism have much wider roles, and that they participate in the neural and hormonal regulation of a variety of intracellular processes.

scholarly journals The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain

2006 ◽  
Vol 27 (5) ◽  
pp. 1581-1591 ◽  
Author(s):  
Pauline Douglas ◽  
Xiaoping Cui ◽  
Wesley D. Block ◽  
Yaping Yu ◽  
Shikha Gupta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Kinase Domain ◽  
Dependent Protein

ABSTRACT The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

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