Phosphorylation of lens intrinsic membrane proteins by protein kinase C

Abstract

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (MI, aPP) of 26000 and 18000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H 1 phosphorylation dependent on calcium and phospholipid but not on CAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at MI, app = 26000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [Y-~'P]ATP showed major bands at MI, app = 18000 and 26000. Several lines of evidence indicated that the label at MI, app = 26000 was associated with MP26, a protein which has been found in lens junctions and which may form cell-cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V 8 protease cleaved the major band at M,, app = 26000 to fragments of M,, app = 22000 and 24000. Label was not,detected in the resulting MI, app = 22000 peptide, but the MI, app = 24000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for CAMP-dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.Lens membranes contain a major intrinsic protein, often referred to as MP26 or MIP26 due to the apparent MI on SDS-PAGE of 26000 [l]. MP26 constitutes approximately a half of the total intrinsic protein of the lens membrane [2]. Although questions remain about the biological role of MP26 [3], evidence indicates it is found within lens intercellular junctions [4] and is capable of forming membrane channels [5-81. The sequence of MP26 has recently been determined from cDNA clones and the authors offer a model for the structure of MP26 within a lipid membrane [9]. This model, based on hydrophobicity analysis and other techniques, is consistent with MP26 monomers associating to form a hydrophilic channel. The relative molecular mass of MP26 is 28200 based on this sequence. Thus, MP26 has several features in common with gap junction proteins considered in a variety of tissues [lo-121.The regulation of gap-junction-dependent communication is not well understood in any tissue. However, it is known that the permeability of junctions between different cells can be reduced by elevated cytoplasmic concentrations of calcium and hydrogen ions [13, 141. In addition, agents or manipulations that elevate the cytoplasmic levels of CAMP have been reported to decrease rapidly and reversibly the Correspondence to

The channel architecture of aquaporin 0 at a 2.2-Å resolution

“…Modulation by changes in pH or ion concentration have been suggested in the control of transport of water and possibly other substrates (10,11,13,(30)(31)(32)(33)(34)(35) through AQP0. However, Boron and coworkers (13) argue against these effects and show zero dependence on pH or Ca 2ϩ .…”

EDTA-extractable proteins from calf lens fiber membranes are phosphorylated by Ca2+-phospholipid-dependent protein kinase

“…Recently Lampe et al (1986) investigated the in vitro phosphorylation of lens membrane proteins by protein kinase C prepared from bovine lens and bovine brain. Ill contrast with our results no significant phosphorylation could be detected in the 30000-38000 EEP MW range on SDS-polyaerylamide-gel patterns of their phosphorylated membranes.…”

“…Ill contrast with our results no significant phosphorylation could be detected in the 30000-38000 EEP MW range on SDS-polyaerylamide-gel patterns of their phosphorylated membranes. In the study of Lampe et al (1986), however, EDTA was present in the homogenization buffers during membrane isolation resulting in a EEP-free membrane preparation (van den Eijnden-van Raaij et al, 1985a;van Raaij et al, 1983b). Although EDTA was added to avoid protease activity no significant proteolytie degradation occurs during the preparation of lens membranes even in l~he presence of calcium (van Raaij et al, 1983b).…”

“…As these proteins are still present in urea-treated membranes prepared in the presence of EGTA they might belong to the group of intrinsically located membrane proteins. Considering the results of Lampe et al, (1986) the 50000-60000 MW proteins might represent aggregates of the main intrinsic protein (MW 26000) as well as the 17500 intrinsic protein. The latter proteins are good substrates for protein kinase C .…”

“…Recently the presence of a Ca "z+-and phospholipid-dependent protein kinase (protein kinase C) in the lens has been described (Lampe, Bazzi, Nelsestuen and Johnson, 1986). Protein kinase C is thought to play an important role in signal transduction for a number of cellular processes (Nishizuka, 1984a, b).…”