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Calcium-dependent phosphorylation of proteins in rabbit ciliary processes
N Yoshimura1, T W Mittag, S M Podos
1Department of Ophthalmology, Mount Sinai School of Medicine, CUNY, NY 10029.
Investigative Ophthalmology & Visual Science
|April 1, 1989
Summary
Calcium activates protein phosphorylation in rabbit ciliary processes, particularly in the particulate fraction. Vimentin (58 kDa) is a major substrate for calcium-calmodulin-dependent protein kinases.
Area of Science:
- Biochemistry
- Cellular Biology
- Ophthalmology
Background:
- Calcium ions (Ca2+) play crucial roles in cellular signaling pathways.
- Protein phosphorylation is a key regulatory mechanism in cellular processes.
- Ciliary processes are involved in aqueous humor production and intraocular pressure regulation.
Purpose of the Study:
- To investigate calcium-dependent phosphorylation of endogenous proteins in albino rabbit ciliary processes.
- To identify specific protein substrates and the kinases involved in calcium-mediated signaling.
- To compare the effects of different calcium activators, including calmodulin (CAM) and phorbol myristate acetate (PMA).
Main Methods:
- SDS-polyacrylamide gel electrophoresis and autoradiography were employed to analyze protein phosphorylation.
- Western blotting using anti-vimentin antibody was used for protein identification.
- Triton-X100 treatment was utilized to characterize protein behavior.
Main Results:
- Calcium significantly enhanced protein phosphorylation, especially in the particulate fraction of ciliary processes.
- Calcium-calmodulin (Ca2+-CAM) dependent phosphorylation was more pronounced than calcium-phorbol myristate acetate (Ca2+-PMA) dependent phosphorylation.
- Vimentin (58 kDa) was identified as a major substrate for Ca2+-CAM-dependent protein kinases, with other substrates identified at various molecular weights.
- Protein kinase C (Ca2+-PMA dependent) phosphorylated proteins at 87 kDa and potentially 56/54 kDa.
- A 50 kDa protein was phosphorylated by Ca2+ alone, and a 43 kDa protein underwent Ca2+-dependent dephosphorylation.
Conclusions:
- Calcium-dependent protein phosphorylation is a significant regulatory mechanism in rabbit ciliary processes.
- Vimentin is a key substrate for Ca2+-CAM-dependent kinases in this tissue.
- Distinct protein substrates are phosphorylated by different calcium-activated kinase pathways, suggesting specialized roles in ciliary function.