scholarly journals Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro.

1982 ◽  
Vol 95 (3) ◽  
pp. 918-923 ◽  
Author(s):  
S D Freedman ◽  
J D Jamieson
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Exocrine Pancreas ◽  
Second Messengers ◽  
Subcellular Fractions ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exocrine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One- and two-dimensional gel analysis of 32Pi-labeled pancreatic and parotid lobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction. To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca2+, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca2+- and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca2+ and/or cAMP.

scholarly journals Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact Cells

1998 ◽  
Vol 140 (1) ◽  
pp. 131-141 ◽  
Author(s):  
Helena Melander Gradin ◽  
Niklas Larsson ◽  
Ulrica Marklund ◽  
Martin Gullberg
Keyword(s):  
Protein Kinase ◽  
Genetic System ◽  
Cell Surface Receptor ◽  
In Vitro Assays ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Camp Dependent Protein Kinase ◽  
Oncoprotein 18 ◽  
Dependent Protein

Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site–deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists.

cAMP-dependent protein kinase mediates hydrosmotic effect of vasopressin in collecting duct

1992 ◽  
Vol 263 (1) ◽  
pp. C147-C153 ◽  
Author(s):  
H. M. Snyder ◽  
T. D. Noland ◽  
M. D. Breyer
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Water Permeability ◽  
Collecting Duct ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Pka Regulatory Subunit

The role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) in mediating the hydrosmotic effect of vasopressin in in vitro microperfused rabbit cortical collecting ducts (CCDs) was examined. We measured PKA substrate phosphorylation and water permeability [hydraulic conductivity (Lp) = 10(-7) cm.atm-1.s-1], stimulated by substituted cAMP analogues selective for a unique cAMP binding site (site A or B) on PKA regulatory subunit (R). Synergy between site A- and site B-selective analogues suggests involvement of PKA, because both sites must be occupied for R to dissociate from the catalytic subunit (C), allowing phosphorylation to proceed. As single agents, the site B-selective analogues 8-(4-chlorophenylthio)-cAMP (8-CPT) and 8-thiomethyl-cAMP (8-SCH3) were at least two orders of magnitude more potent than the site A-selective analogues N6-monobutyryl-cAMP (N6-mono) or N6-benzoyl-cAMP (N6-benz). Combinations of subthreshold concentrations of two site A analogues (N6-mono+N6-benz) or two site B-selective analogues (8-CPT + 8-SCH3) failed to significantly increase protein phosphorylation or water permeability. In contrast, combination of a site A plus site B analogue synergistically stimulated both protein phosphorylation and Lp. Rp-cAMPS, an inhibitor of cAMP binding to PKA, reduced both vasopressin (41% inhibition)- and cAMP (56% inhibition)-stimulated water permeability. H-89 (50 microM), an inhibitor of PKA kinase activity, also blocked cAMP-stimulated water permeability (90% inhibition). These findings suggest that vasopressin-induced water permeability in the rabbit CCD is mediated by PKA.

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.

scholarly journals Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase

2002 ◽  
Vol 368 (1) ◽  
pp. 243-251 ◽  
Author(s):  
Pauline DOUGLAS ◽  
Gopal P. SAPKOTA ◽  
Nick MORRICE ◽  
Yaping YU ◽  
Aaron A. GOODARZI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Activity ◽  
Catalytic Subunit ◽  
Protein Sequencing ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

scholarly journals Enzymic characteristics of an ecto-cyclic AMP-dependent protein kinase in rat epididymal spermatozoa

1981 ◽  
Vol 195 (1) ◽  
pp. 111-117 ◽  
Author(s):  
G C Majumder
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
External Surface ◽  
Cell Damage ◽  
Surface Protein ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

Intact spermatozoa from rat cauda epididymides possess an ecto-(cyclic AMP-dependent protein kinase) activity that causes the transfer of the terminal phosphate group of ATP to the serine residues of all the histone fractions. The enzyme showed a high degree of substrate specificity for the phosphorylation of histones rather than protamine, casein and phosvitin. The cell-external-surface protein kinase requires Mg2+ for activity, and other bivalent cations such as Mn2+ and Co2+ can substitute partially for Mg2+, whereas Ca2+ and Zn2+ are potent inhibitors of the enzyme. The enzyme has markedly higher affinity for cyclic AMP than for other cyclic nucleotides for its activation, with an apparent Km value for cyclic AmP of 80 nM. Spermatozoal ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. There was no loss of kinase activity from the cells when washed with 2 mM-EDTA, and the histones phosphorylated by intact spermatozoa were located outside the cells. Protein kinase activity of intact cells was strongly inhibited (approx. 90%) by p-chloromercuribenzenesulphonic acid (10 microM), which is believed not to enter the cells. These data provide further support for the localization of a protein kinase on the external surface of spermatozoa.

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