A lens intercellular junction protein, MP26, is a phosphoprotein

K. R. Johnson, P. D. Lampe, K. C. Hur, C. F. Louis

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at M(r) 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at M(r) 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at M(r) 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated M(r) 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within ~20 to 40 residues from the COOH-terminus of MP26. Published work indicates that phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.

Original languageEnglish (US)
Pages (from-to)1334-1343
Number of pages10
JournalJournal of Cell Biology
Volume102
Issue number4
DOIs
StatePublished - Jan 1 1986

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Intercellular Junctions
Phosphoproteins
Lenses
Proteins
Crystallins
Serine
Membranes
Phosphorylation
Cell Membrane
Phosphoamino Acids
Connexins
Colforsin
Threonine
Cyclic AMP-Dependent Protein Kinases
Autoradiography
Detergents
Tyrosine
Urea
Polyacrylamide Gel Electrophoresis
Membrane Proteins

ASJC Scopus subject areas

  • Cell Biology

Cite this

A lens intercellular junction protein, MP26, is a phosphoprotein. / Johnson, K. R.; Lampe, P. D.; Hur, K. C.; Louis, C. F.

In: Journal of Cell Biology, Vol. 102, No. 4, 01.01.1986, p. 1334-1343.

Research output: Contribution to journalArticle

Johnson, K. R. ; Lampe, P. D. ; Hur, K. C. ; Louis, C. F. / A lens intercellular junction protein, MP26, is a phosphoprotein. In: Journal of Cell Biology. 1986 ; Vol. 102, No. 4. pp. 1334-1343.
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abstract = "The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at M(r) 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at M(r) 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at M(r) 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated M(r) 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within ~20 to 40 residues from the COOH-terminus of MP26. Published work indicates that phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10{\%} threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.",
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