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A major, transformation-sensitive PKC-binding protein is also a PKC substrate involved in cytoskeletal remodeling
C Chapline1, J Cottom, H Tobin
1Adirondack Biomedical Research Institute, Lake Placid, New York 12946, USA.
The Journal of Biological Chemistry
|July 25, 1998
Summary
This study identifies clone 72 as a direct substrate of Protein Kinase C (PKC), demonstrating its phosphorylation in cells and involvement in membrane remodeling.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Protein Kinase C (PKC) regulates critical cellular processes like growth and gene expression.
- Identifying specific PKC-mediated phosphorylation events has been challenging.
- A major PKC-binding protein, clone 72, was previously identified in REF52 fibroblasts.
Purpose of the Study:
- To determine if clone 72 is a direct substrate of PKC.
- To identify the specific phosphorylation sites on clone 72.
- To investigate the cellular localization and function of phosphorylated clone 72.
Main Methods:
- In vitro and in vivo phosphorylation assays.
- Phosphopeptide mapping, Edman degradation, and mass spectrometry to identify phosphorylation sites.
- Development of phosphorylation state-selective antibodies for cellular analysis.
Main Results:
- Clone 72 is phosphorylated by PKC both in vitro and in vivo.
- Serine residues 283, 300, 507, and 515 were identified as major PKC phosphorylation sites.
- Phosphorylated clone 72 accumulates in membrane protrusions and ruffles upon PKC activation.
Conclusions:
- Clone 72 is a direct substrate of PKC, phosphorylated at specific serine residues.
- PKC-mediated phosphorylation of clone 72 is involved in membrane-cytoskeleton remodeling.
- These findings support the model of PKC interacting with and modifying substrate proteins (STICKs).