Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay

M. P. Lutz, D. I. Pinon, Laurence J Miller

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.

Original languageEnglish (US)
Pages (from-to)418-424
Number of pages7
JournalPancreas
Volume9
Issue number4
StatePublished - 1994

Fingerprint

Phosphoprotein Phosphatases
Phosphoric Monoester Hydrolases
Pancreas
Cell Membrane
Cytosol
Enzymes
Phosphorylase a
Cholecystokinin Receptors
Protein Phosphatase 1
Protein Phosphatase 2
Acinar Cells
Calcineurin
Phosphoproteins
Phosphorylation
Proteins

Keywords

  • Cholecystokinin receptor
  • Phosphorylation
  • Protein phosphatase

ASJC Scopus subject areas

  • Endocrinology
  • Gastroenterology

Cite this

Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay. / Lutz, M. P.; Pinon, D. I.; Miller, Laurence J.

In: Pancreas, Vol. 9, No. 4, 1994, p. 418-424.

Research output: Contribution to journalArticle

@article{db2a83a21cd2473da35e6d69353d7d82,
title = "Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay",
abstract = "Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.",
keywords = "Cholecystokinin receptor, Phosphorylation, Protein phosphatase",
author = "Lutz, {M. P.} and Pinon, {D. I.} and Miller, {Laurence J}",
year = "1994",
language = "English (US)",
volume = "9",
pages = "418--424",
journal = "Pancreas",
issn = "0885-3177",
publisher = "Lippincott Williams and Wilkins",
number = "4",

}

TY - JOUR

T1 - Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay

AU - Lutz, M. P.

AU - Pinon, D. I.

AU - Miller, Laurence J

PY - 1994

Y1 - 1994

N2 - Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.

AB - Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.

KW - Cholecystokinin receptor

KW - Phosphorylation

KW - Protein phosphatase

UR - http://www.scopus.com/inward/record.url?scp=0028359611&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028359611&partnerID=8YFLogxK

M3 - Article

VL - 9

SP - 418

EP - 424

JO - Pancreas

JF - Pancreas

SN - 0885-3177

IS - 4

ER -