Cyclic AMP-mediated phosphorylation and insolubilization of a 49-kDa cytoskeletal marker protein of lens fiber terminal differentiation.
Terminal differentiation of chick lens fiber cells has been previously characterized by the accumulation, acidification via phosphorylation, and increased membrane association of a 49-kDa cytoskeletal protein. In these studies, we examine: (1) the subcellular distribution of the 49-kDa protein with regard to ageing and isoform composition; and (2) potential mechanisms regulating 49-kDa phosphorylation and insolubilization. With conventional Western blotting techniques, the 49-kDa protein is found exclusively in insoluble form within terminally differentiated nuclear fiber cells. Cortical fibers, on the other hand, exhibit a more widespread subcellular distribution of the 49-kDa protein. On two-dimensional gels, cortical 49-kDa isoelectric variants segregate according to their ease of sedimentation. After homogenization in detergent-containing buffers, the major isoform of the 49-kDa protein found in low speed pellets (40,000 g, 20 min) exhibits an acidic pI. The 40,000 g supernate and the high speed pellet (100,000 g, 2 hr) which is sedimented from this supernate are enriched in more basic isoforms of the 49-kDa protein. The 100,000 g supernate overlying the high speed pellet is dominated by the most basic isoform. With in vitro phosphorylation assays, the 49 kDa protein is shown to be a major substrate affected by endogenous cAMP-dependent mechanisms. Both the low and high speed pellets exhibit endogenous cAMP-dependent kinase activity. An inhibitor of cAMP-dependent protein kinase activity is also found in soluble lens fractions. Conversion of the 49-kDa protein into more acidic, phosphorylated isoforms increases its insolubility and ease of sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)
Ireland
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ME
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201.
Klettner
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C
Nunlee
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D000375:Aging / Q000502:physiology; D000818:Animals; D000943:Antigens, Differentiation; D002454:Cell Differentiation; D002645:Chickens; D000242:Cyclic AMP / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007904:Lens Cortex, Crystalline / Q000166:cytology*; D008970:Molecular Weight; D010766:Phosphorylation / Q000187:drug effects; D012995:Solubility
D000943:Antigens, Differentiation; D003598:Cytoskeletal Proteins; D000242:Cyclic AMP