Linkage of a nucleolin-related protein and casein kinase II with the detergent-stable photoreceptor cytoskeleton.
Vertebrate photoreceptors are highly polarized sensory neurons with a complex microtubule and actin-based cytoskeletal organization. In the present study, we have used a detergent-extracted cytokeleton preparation from bovine photoreceptors to test the hypothesis that protein kinases and their substrates co-purify with the photoreceptor cytoskeleton. We incubated the cytoskeletal preparation in the presence of [gamma-32P]ATP. Following SDS-PAGE and autoradiography, we found two principal phosphoproteins with apparent molecular weights of 55 kDa (pp55) and 112 kDa (pp112). We have additionally identified the kinase responsible for phosphorylation of pp112 (and possibly pp55) as a casein kinase II-like enzyme. pp55 was identified as beta-tubulin based on Western blotting and its position on two-dimensional gels. Microsequencing revealed that 16 of the first 17 amino acids of pp112 were identical to human nucleolin, a nuclear protein. Western blotting, mobility in SDS PAGE and in two-dimensional gels, predominant localization within the nucleus, and phosphorylation by a casein kinase II all support the conclusion that pp112 is a nucleolin-related protein. Immunocytochemistry revealed a significant extranuclear pool of nucleolin-immunoreactivity within the cell bodies of photoreceptors. These findings suggest an important extranuclear role for nucleolin or a related protein in photoreceptors.
Hollander
B A
BA
Department of Anatomy and Cell Biology, The University of Kansas Medical Center, Kansas City, USA.
Liang
M Y
MY
Besharse
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D002417:Cattle; D002458:Cell Fractionation; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D017830:Octoxynol; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000032:analysis; D000097796:Nucleolin
D047390:Casein Kinase II; D003598:Cytoskeletal Proteins; D003902:Detergents; D009687:Nuclear Proteins; D017830:Octoxynol; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D016601:RNA-Binding Proteins; D014404:Tubulin; D000097796:Nucleolin