Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

Keeling, Peggy; Johnson, Keith R.; Sas, Daryl F.; Klukas, Kathleen A.; Donahue, P. N.; Johnson, Ross G.

doi:10.1007/bf02332125

Cited by 41 publications

(30 citation statements)

References 36 publications

(49 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The limited cleavage of MP26 observed here after incubation of membranes with chymotrypsin, trypsin, or V8 protease is consistent with previous reports (27,28). In each case, there is a total loss of 4-5 kDa primarily from the carboxyl terminus.…”

Section: Discussionsupporting

confidence: 92%

“…Several minor bands are always present in these preparations, and some of these are more abundant in membranes prepared from the nuclear region of the lens. One minor band that migrates at a position of 19-20 kDa is consistently present in these preparations and those of others (18 kDa) (10,23,(27)(28)(29)(30). When electrophoresis was performed using gradient gels, these proteins migrated at 28-29 kDa and at 19-22 kDa, respectively.…”

Section: Resultssupporting

confidence: 63%

See 1 more Smart Citation

Phosphorylation of lens fiber cell membrane proteins.

Garland¹,

Russell²

1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Two intrinsic membrane proteins of calf lens fiber cells can be phosphorylated by a soluble bovine lens cAMP-dependent protein kinase and rabbit muscle cAMP-dependent protein kinase. After electrophoresis of the phosphorylated membranes, 32p comigrates with the lens main intrinsic protein at 26-27 kDa and with a minor band of protein that migrates at 19-20 kDa. 32p is also found with proteins that, based on the molecular sizes, are likely multimers of the 19-kDa and 26-kDa proteins. Upon boiling in NaDodSO4, all the radioactivity is found at the top of the gel, suggesting that both phosphoproteins are intrinsic membrane proteins. Serine is the only phospho amino acid detected in both proteins regardless of the source of protein kinase. Recent studies have demonstrated a link between disruption of lens fiber membranes and formation of cataracts (1-5). As a result, there is increased interest in the structure and function of lens membranes.Numerous studies have elucidated the composition of the lens fiber cell membrane (6-13). One protein of 26-28 kDa (MP26) constitutes '50% of the intrinsic proteins of these membranes. Several minor species, including one of [18][19][20] kDa, also constitute the intrinsic membrane proteins. Approximately 60% of the fiber cell membrane is involved in the intercellular communicating junctions, commonly referred to as gap junctions. Since MP26 is the most abundant protein in the fiber membranes, it is probably the major protein in these regions (8-10). MP26 is localized in and outside the junctional regions (14-16). This suggests that, in addition to some specialized function in the gap junction, the MP26 protein may play another role.In tissues other than the lens there is considerable evidence that intercellular communication is mediated by the opening and closing of gap junctions in response to variations in the concentration of calcium (17)(18)(19)(20)(21)(22). In the lens, however, it is thought that the junctions are always open and are, therefore, insensitive to environmental stimuli such as the calcium level (23). Nevertheless, recent studies suggest that the uncoupling of cell communication in the lens can be provoked by shifts in the calcium concentration (24). In view of the fact that the activity of some proteins (enzymes) is altered by mechanisms involving phosphorylation, the present study was undertaken to explore the possibility that the phosphorylation of the MP26 protein might be involved in its biological function. In this report, we describe results of studies showing that the phosphorylation of two intrinsic membrane proteins is catalyzed by an endogenous lens cAMP-dependent protein kinase and a cAMP-dependent protein kinase from rabbit muscle. MATERIALS AND METHODS Preparation of Lens Fiber Cell Membranes and LensProtein Kinase. Lens fiber cell membranes were prepared from 4-month-old calf lenses according to the procedure of Russell et al. (25). Bovine lens protein kinase was prepared from lens cortex as follows: extracts were prepared by homogenizing ...

show abstract

Section: Discussionsupporting

confidence: 92%

Section: Resultssupporting

confidence: 63%

Phosphorylation of lens fiber cell membrane proteins.

Garland¹,

Russell²

1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This conclusion is consistent with a number of other studies whch suggested that proteases remove a C-terminal piece with M , = 4000 -5000 from the cytoplasmic side of the lens membrane [40,41]. A less prominent intermediate at M,, app = 25500 (lanes 2, 3) was also present as previously observed [40]. The M,, app = 18000 protein was not as susceptible to chymotrypsin cleavage and did not appear to be degraded as thoroughly in the reaction time course.…”

Section: Properties Of Protein Kinase C Preparationssupporting

confidence: 93%

“…The kinetics of these changes and the similar staining intensities suggested that the M,, app = 22000 protein was a cleavage product of MP26. This conclusion is consistent with a number of other studies whch suggested that proteases remove a C-terminal piece with M , = 4000 -5000 from the cytoplasmic side of the lens membrane [40,41]. A less prominent intermediate at M,, app = 25500 (lanes 2, 3) was also present as previously observed [40].…”

Section: Properties Of Protein Kinase C Preparationssupporting

confidence: 92%

Phosphorylation of lens intrinsic membrane proteins by protein kinase C

Lampe¹,

Bazzi

Nelsestuen

et al. 1986

Eur J Biochem

View full text Add to dashboard Cite

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

show abstract

“…3B). Inspection of this figure indicates some 32P is also associated with the 24-kDa componcnt that is derived from the chymotryptic cleavage of some larger lens membrane component [24]. Fig.…”

Section: Resultsmentioning

confidence: 96%

Characterization of the bovine lens plasma membrane substrates for cAMP‐dependent protein kinase

Louis¹,

Johnson

et al. 1985

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

CAMP-dependent protein kinase, derived from either calf lens or bovine heart, promotes the phosphorylation of three lens plasma membrane proteins of molecular mass 28 kDa, 26 kDa and 18 kDa. Correlation of the maximal level of phosphorylation of these components with the Coomassie blue staining intensity of fractionated lens membranes suggests that the phosphorylation of the 28 kDa and 18 kDa components may be approximately stoichiometric. The protein kinasc substrates could be dephosphorylated by a cardiac sarcoplasmic-reticulumbound protein phosphatase activity. The 26 kDa component comigrated with MP26, the major lens membrane component that has been localized to the lens fiber cell junction. Treatment of phosphorylated lens membranes with chymotrypsin did not suggest that any of the three major phosphorylated components was derived from the partial proteolysis of a larger phosphoprotein. After electrophoretic separation of phosphorylated proteins, treatment with N-chlorosuccinimide confirmed that there was little similarity in the structure of the three phosphoproteins. Chymotrypsin did, however, reveal a cryptic phosphorylation site in a 22 kDa fragment that appeared to be derived from MP26. Treatment of phosphorylated membranes with reducing agents resulted in the disappearance of the 28 kDa phosphorylated component and the appearance of a new phosphorylated component of 18 kDa; neither MP26 nor the original 38 kDa component was affected by such treatment. It is not clear whether the original 18 kDa phosphoprotein, present in unreduced samples, is the same as that generated with reducing agents from the 28 kDa phosphorylated lens membrane component.

show abstract

Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

Cited by 41 publications

References 36 publications

Phosphorylation of lens fiber cell membrane proteins.

Phosphorylation of lens fiber cell membrane proteins.

Phosphorylation of lens intrinsic membrane proteins by protein kinase C

Characterization of the bovine lens plasma membrane substrates for cAMP‐dependent protein kinase

Contact Info

Product

Resources

About