A Green Fluorescent Protein Kinase Substrate Allowing Detection and Localization of Intracellular ERK/MAP Kinase Activity

2001 ◽  
Vol 293 (2) ◽  
pp. 264-268 ◽  
Author(s):  
James W. Mandell ◽  
Nigel C. Gocan
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Kinase ◽  
Map Kinase ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Kinase Substrate ◽  
Green Fluorescent ◽  
Detection And Localization

scholarly journals The Lipophilicity of Phorbol Esters as a Critical Factor in Determining the Pattern of Translocation of Protein Kinase C δ Fused to Green Fluorescent Protein

2000 ◽  
Vol 275 (16) ◽  
pp. 12136-12146 ◽  
Author(s):  
Qiming J. Wang ◽  
Tzan-Wei Fang ◽  
David Fenick ◽  
Susan Garfield ◽  
Bruno Bienfait ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Fluorescent Protein ◽  
Critical Factor ◽  
Kinase C ◽  
Green Fluorescent

scholarly journals Pak1 Protein Kinase Regulates Activation and Nuclear Localization of Snf1-Gal83 Protein Kinase

2004 ◽  
Vol 24 (18) ◽  
pp. 8255-8263 ◽  
Author(s):  
Kristina Hedbacker ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Kinase Activity ◽  
Activation Loop ◽  
Glucose Limitation ◽  
Subunit Isoforms ◽  
Green Fluorescent ◽  
Amp Activated Protein Kinase

ABSTRACT Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the β-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Δ mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

scholarly journals Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

2006 ◽  
Vol 1763 (4) ◽  
pp. 337-344 ◽  
Author(s):  
Chisato Kosugi-Tanaka ◽  
Xuefei Li ◽  
Chenjuan Yao ◽  
Tetsuya Akamatsu ◽  
Norio Kanamori ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Aquaporin 5 ◽  
Green Fluorescent ◽  
Kinase A

scholarly journals Regulation of ZAP-70 Intracellular Localization: Visualization with the Green Fluorescent Protein

1997 ◽  
Vol 186 (10) ◽  
pp. 1713-1724 ◽  
Author(s):  
Joanne Sloan-Lancaster ◽  
Weiguo Zhang ◽  
John Presley ◽  
Brandi L. Williams ◽  
Robert T. Abraham ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
T Cell Receptor ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Cell Receptor ◽  
Cellular Activation ◽  
Intracellular Location ◽  
Green Fluorescent ◽  
Biologic Function

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

scholarly journals 1144-128 Calmodulin kinase activity links green fluorescent protein overexpression to a cardiomyopathic phenotype

2004 ◽  
Vol 43 (5) ◽  
pp. A219
Author(s):  
Michelle S Khoo ◽  
Rong Zhang ◽  
Chad E Grueter ◽  
Gemin Ni ◽  
Martha A Bass ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Overexpression ◽  
Calmodulin Kinase ◽  
Green Fluorescent

scholarly journals Differential Localization of Protein Kinase C δ by Phorbol Esters and Related Compounds Using a Fusion Protein with Green Fluorescent Protein

1999 ◽  
Vol 274 (52) ◽  
pp. 37233-37239 ◽  
Author(s):  
Qiming J. Wang ◽  
Dipak Bhattacharyya ◽  
Susan Garfield ◽  
Kassoum Nacro ◽  
Victor E. Marquez ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Phorbol Esters ◽  
Fluorescent Protein ◽  
Kinase C ◽  
Differential Localization ◽  
Green Fluorescent ◽  
Related Compounds

scholarly journals Three Distinct Mechanisms for Translocation and Activation of the δ Subspecies of Protein Kinase C

1998 ◽  
Vol 18 (9) ◽  
pp. 5263-5271 ◽  
Author(s):  
Shiho Ohmori ◽  
Yasuhito Shirai ◽  
Norio Sakai ◽  
Motoko Fujii ◽  
Hiroaki Konishi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Simultaneous Treatment ◽  
Kinase C ◽  
Green Fluorescent ◽  
Number Of Cells

ABSTRACT We expressed δ subspecies of protein kinase C (δ-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The δ-PKC–GFP fusion protein had enzymological characteristics very similar to those of the native δ-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of δ-PKC–GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM), induced a slower translocation of δ-PKC–GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of δ-PKC–GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated δ-PKC–GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate δ-PKC–GFP but increased its kinase activity more than threefold. δ-PKC–GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of δ-PKC–GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated δ-PKC–GFP up to more than fivefold. TPA treatment of cells overexpressing δ-PKC–GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of δ-PKC.

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