scholarly journals Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells

1992 ◽  
Vol 205 (2) ◽  
pp. 471-481 ◽  
Author(s):  
Martin EIGENTHALER ◽  
Christine NOLTE ◽  
Maria HALBRUGGE ◽  
Ulrich WALTER
Keyword(s):  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Human Platelets ◽  
Intact Cells ◽  
Dependent Protein

Possible Roles for Protein Phosphorylation in Platelet Responses

1979 ◽  
Author(s):  
R.J. Haslam ◽  
J.E.B. Fox ◽  
S.E. Salama ◽  
J.A. Lynham
Keyword(s):  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Platelet Function ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Type I ◽  
Sds Page ◽  
Dependent Protein ◽  
32P Incorporation

The relationships between the phosphorylation of specific platelet polypeptides and platelet function were studied using washed human platelets labelled by preincubation with [32p] Pi. Platelet polypeptides were separated by SDS-PAGE and 32P incorporation into them determined by autoradiography. Whereas induction of platelet aggregation alone did not affect protein phosphorylation, induction of the release reaction increased 3P incorporation into several polypeptides (P75,P47,P40,P27,P20,P19), including the P-light chain of platelet myosin (P20). These changes were inhibited by drugs that blocked Ca2 movements and may be due to activation of Ca2+-dependent protein kinases. Compounds that inhibited platelet function by increasing cyclic AMP (e.g. PCE1) also suppressed these reactions but, in addition, increased phosphorylation of other polypeptides (P50,P49,P36,P24,P22). Type I and Type II cyclic AMP-dependent protein kinases were present in platelets and may mediate Che latter effects of cyclic AMP. Subcellular fractionation of 32p-labelled platelets that had been exposed to PCE1 showed that P24 was present in membranes that could take up Ca2+ by an ATP-dependent mechanism. Membranes from PCE1-treated platelets took up Ca2+ more rapidly than control membranes. Thus, the cyclic AMP-dependent phosphorylation of P24 may stimulate the removal of Ca2+ from platelet cytosol and suppress Ca2+-dependent phosphorylation reactions necessary for release of granule constituents.

scholarly journals Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.

1980 ◽  
Vol 87 (2) ◽  
pp. 336-345 ◽  
Author(s):  
C L Browne ◽  
A H Lockwood ◽  
J L Su ◽  
J A Beavo ◽  
A L Steiner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Cyclic Nucleotides ◽  
Mitotic Spindle ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Mitotic Apparatus ◽  
Dependent Protein

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.

scholarly journals Role of cyclic nucleotides in store-mediated external Ca2+ entry in human platelets

1995 ◽  
Vol 310 (1) ◽  
pp. 263-269 ◽  
Author(s):  
K Nakamura ◽  
M Kimura ◽  
A Aviv
Keyword(s):  
Cyclic Amp ◽  
Tyrosine Kinase ◽  
Sodium Nitroprusside ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Cyclic Nucleotides ◽  
Human Platelets ◽  
Dependent Protein ◽  
Kinase Phosphorylation

This study explores the role of cyclic nucleotides (i.e. cyclic AMP and cyclic GMP) in store-regulated external Ca2+ entry in human platelets. To stimulate store-regulated external Ca2+ entry, thapsigargin was used to deplete Ca2+ from the dense tubules, and sodium nitroprusside and iloprost respectively were used to stimulate endogenous cyclic GMP and cyclic AMP formation. Pretreatment with sodium nitroprusside and iloprost (a) attenuated the thapsigargin-evoked external Ca2+ entry and (b) reduced the rate of Ca2+ release from the dense tubules. The effects on external Ca2+ entry and Ca2+ release from the dense tubules were exerted independently and were apparently mediated through activation of the respective cyclic nucleotide-dependent protein kinases. Both sodium nitroprusside and iloprost reduced tyrosine kinase phosphorylation of a number of proteins, particularly a 72 kDa protein band. Both agents also attenuated the thapsigargin-evoked tyrosine kinase phosphorylation of the 72 kDa band. Intracellular Ca2+ depletion resulted in a reduction in tyrosine kinase-mediated phosphorylation of a number of protein bands, including the 72 kDa band and the further attenuation of thapsigargin-mediated tyrosine phosphorylation of this band. The effects of the cyclic nucleotides on cellular Ca2+ homoeostasis in thapsigargin-treated platelets were not exerted via acceleration of Ca2+ extrusion or Ca2+ sequestration into the mitochondria. We conclude that cyclic nucleotides participate in store-regulated control of external Ca2+ entry by slowing down the rate of external Ca2+ entry and Ca2+ release from intracellular Ca2+ stores. These effects are apparently mediated via cyclic nucleotide-dependent protein kinases and the attenuation of protein phosphorylation by tyrosine kinases.

IN VITRO ASSAYS OF ADENYL CYCLASE

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S337-S347 ◽  
Author(s):  
Martin Rodbell
Keyword(s):  
Protein Kinases ◽  
Cyclic Nucleotide ◽  
Current Knowledge ◽  
Adenyl Cyclase ◽  
In Vitro Assays ◽  
Animal Cells ◽  
Intact Cells ◽  
Dependent Protein

ABSTRACT A brief review of the properties and current knowledge of the components of adenyl cyclase systems in animal cells is presented, followed by some general remarks on the problems of assaying adenyl cyclase. Recent techniques developed for the assay of adenyl cyclase in broken cell preparations are described. Methods which determine the levels of cyclic 3′5′AMP in intact cells or tissues are also presented, with particular emphasis on the use of binding of the nucleotide to cyclic 3′5′AMP dependent protein kinases and radioimmunoassays of the cyclic nucleotide.

scholarly journals Cyclic nucleotide-dependent protein kinases in airway smooth muscle.

1982 ◽  
Vol 257 (19) ◽  
pp. 11609-11616 ◽  
Author(s):  
T J Torphy ◽  
W B Freese ◽  
G A Rinard ◽  
L L Brunton ◽  
S E Mayer
Keyword(s):  
Smooth Muscle ◽  
Protein Kinases ◽  
Airway Smooth Muscle ◽  
Cyclic Nucleotide ◽  
Dependent Protein
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