TITLE

Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis

AUTHOR(S)
Rauf, Femina; Huang, Yiding; Muhandiramlage, Thusitha; Aspinwall, Craig
PUB. DATE
August 2010
SOURCE
Analytical & Bioanalytical Chemistry;Aug2010, Vol. 397 Issue 8, p3359
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
A cell-penetrating, fluorescent protein substrate was developed to monitor intracellular protein kinase A (PKA) activity in cells without the need for cellular transfection. The PKA substrate (PKAS) was prepared with a 6×histidine purification tag, an enhanced green fluorescent protein (EGFP) reporter, an HIV-TAT protein transduction domain for cellular translocation and a pentaphosphorylation motif specific for PKA. PKAS was expressed in Escherichia coli and purified by metal affinity chromatography. Incubation of PKAS in the extracellular media facilitated translocation into the intracellular milieu in HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity in a time and concentration dependent manner. Upon cellular loading, glucose-dependent phosphorylation of PKAS was observed in both βTC-3 cells and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6%) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4%) in βTC-3 cells was observed at 3 mM glucose indicating a left-shifted glucose sensitivity. Increased PKAS phosphorylation was observed in the presence of PKA stimulators forskolin and 8-Br-cAMP (33% and 16%, respectively), with corresponding decreases in PKAS phosphorylation observed in the presence of PKA inhibitors staurosporine and H-89 (40% and 54%, respectively). [Figure not available: see fulltext.]
ACCESSION #
52532533

 

Related Articles

  • β-Cell-Specific Protein Kinase A Activation Enhances the Efficiency of Glucose Control by Increasing Acute-Phase Insulin Secretion. Kaihara, Kelly A.; Dickson, Lorna M.; Jacobson, David A.; Tamarina, Natalia; Roe, Michael W.; Philipson, Louis H.; Wicksteed, Barton // Diabetes;May2013, Vol. 62 Issue 5, p1527 

    Acute insulin secretion determines the efficiency of glucose clearance. Moreover, impaired acute insulin release is characteristic of reduced glucose control in the prediabetic state. Incretin hormones, which increase β-cell cAMP, restore acute-phase insulin secretion and improve glucose...

  • Effects of High Glucose Levels and Glycated Serum on GIP Responsiveness in the Pancreatic Beta Cell Line HIT-T15. Puddu, Alessandra; Sanguineti, Roberta; Montecucco, Fabrizio; Viviani, Giorgio Luciano // Journal of Diabetes Research;6/29/2015, Vol. 2015, p1 

    Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone produced in the gastrointestinal tract that stimulates glucose dependent insulin secretion. Impaired incretin response has been documented in diabetic patients and was mainly related to the inability of the pancreatic beta...

  • TECHNIQUE: Light up your kinase. Gagescu, Raluca // Nature Reviews Molecular Cell Biology;Feb2002, Vol. 3 Issue 2, p82 

    Deals with a class of genetically encoded fluorescent probes that might allow the understanding of the activation of virtually any kinase in living cells. Characteristics of the probes; Result that occurs if the substrate domain is phosphorylated on the activation of the kinase; Situation when...

  • Increased Intracellular Calcium Is Required for Spreading of Rat Islet Beta-Cells on... Bosco, Domenico; Gonelle-Gispert, Carmen; Wollheim, Claes B.; Halban, Philippe A.; Rouiller, Dominique G. // Diabetes;May2001, Vol. 50 Issue 5, p1039 

    Investigates the role of increased intracellular free calcium concentration as an intracellular step linking glucose stimulation and beta-cell spreading. Spread of rat islet beta- cells in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line;...

  • Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2. Lee, Sheeyong; Duhé, Roy J. // Journal of Biomedical Science;Nov2006, Vol. 13 Issue 6, p773 

    Janus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP)...

  • Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.). Wanwan Liang; Bo Yang; Bao-Jun Yu; Zili Zhou; Cui Li; Ming Jia; Yun Sun; Yue Zhang; Feifei Wu; Hanfeng Zhang; Boya Wang; Deyholos, Michael K.; Yuan-Qing Jiang // BMC Genomics;2013, Vol. 14 Issue 1, p1 

    Background: Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions....

  • Fascin 2b Is a Component of Stereocilia that Lengthens Actin-Based Protrusions. Shih-Wei Chou; Philsang Hwang; Gomez, Gustavo; Fernando, Carol A.; West, Megan C.; Pollock, Lana M.; Lin-Jones, Jennifer; Burnside, Beth; McDermott Jr., Brian M. // PLoS ONE;2011, Vol. 6 Issue 4, p1 

    Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling...

  • Angiotensin II diminishes the effect of SGK1 on the WNK4-mediated inhibition of ROMK1 channels. Peng Yue; Peng Sun; Dao-Hong Lin; Chunyang Pan; Wenming Xing; WenHui Wang // Kidney International;Feb2011, Vol. 79 Issue 4, p423 

    ROMK1 channels are located in the apical membrane of the connecting tubule and cortical collecting duct and mediate the potassium secretion during normal dietary intake. We used a perforated whole-cell patch clamp to explore the effect of angiotensin II on these channels in HEK293 cells...

  • Mitochondrial Networking Protects β-Cells From Nutrient-Induced Apoptosis. Molina, Anthony J. A.; Wikstrom, Jakob D.; Stiles, Linsey; Las, Guy; Mohamed, Hibo; Elorza, Alvaro; Walzer, Gil; Twig, Gilad; Katz, Steve; Corkey, Barbara E.; Shirihaix, Orian S. // Diabetes;Oct2009, Vol. 58 Issue 10, p2303 

    OBJECTIVE--Previous studies have reported that β-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating β-cell mitochondrial morphology and function, remains unclear. In this article, we...

Share

Read the Article

Courtesy of VIRGINIA BEACH PUBLIC LIBRARY AND SYSTEM

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics