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Fragmin, a microfilament regulatory protein from Physarum polycephalum, is phosphorylated by casein kinase II-type
V De Corte1, J Gettemans, Y De Ville
1Flanders Interuniversity Institute for Biotechnology, Laboratory for Physiological Chemistry, University of Ghent, Belgium.
Abstract:
Fragmin is a 42 kDa regulatory protein involved in actin microfilament organization in Physarum polycephalum. We show that fragmin is a target of casein kinase II (CK II) enzymes isolated from evolutionarily divergent species. In Physarum microplasmodia, two such kinases were identified. A serine residue located in the sequence Gly-Gly-Ser-Asp-Leu-Glu constitutes the phosphorylation site and was identified by phosphopeptide sequencing, mass spectometry analysis, and inhibition studies with a synthetic peptide corresponding to this site. Interestingly, the actin-fragmin dimer (A--F) as well as the actin2-fragmin trimer (A2--F) are equally efficient targets, and phosphorylation had no effect on the actin-binding properties of fragmin. Actin-fragmin isolated from microplasmodia revealed a minor acidic fragmin isoform, suggesting that fragmin is phosphorylated in vivo. The actin-fragmin complex is also phosphorylated on the actin subunit by an endogenous actin-fragmin kinase [Gettemans, J., De Ville, Y., Vandekerckhove, J., & Waelkens, E. (1992) EMBO J. 11, 3185-3191]. We show that the two phosphorylation events act independently of each other.
Insights
Fragmin, a regulatory protein in Physarum polycephalum, is phosphorylated by casein kinase II (CK II) enzymes. This phosphorylation occurs independently of actin binding and does not affect fragmin
Area of Science:
- Cell Biology
- Biochemistry
- Protein Kinases
Background:
- Fragmin is a 42 kDa regulatory protein crucial for actin microfilament organization in Physarum polycephalum.
- Actin-binding proteins like fragmin are essential for cellular structure and dynamics.
Purpose of the Study:
- To investigate whether fragmin is a substrate for casein kinase II (CK II) enzymes.
- To identify the specific phosphorylation site on fragmin and its functional implications.
- To determine the relationship between fragmin phosphorylation and its interaction with actin.
Main Methods:
- Isolation and characterization of casein kinase II (CK II) enzymes from Physarum polycephalum.
- Phosphopeptide sequencing and mass spectrometry to identify the phosphorylation site.
- Enzyme inhibition studies using synthetic peptides to confirm the phosphorylation site.
Main Results:
- Fragmin is identified as a direct target of CK II enzymes from evolutionarily divergent species.
- Phosphorylation occurs at a specific serine residue within the Gly-Gly-Ser-Asp-Leu-Glu sequence.
- Phosphorylation of fragmin does not alter its actin-binding properties, affecting neither the actin-fragmin dimer nor the actin2-fragmin trimer.
- Evidence suggests in vivo phosphorylation of fragmin, indicated by the presence of an acidic fragmin isoform.
- Fragmin phosphorylation by CK II and actin phosphorylation by an endogenous kinase occur independently.
Conclusions:
- Fragmin is a substrate for casein kinase II (CK II), with phosphorylation occurring at a distinct serine residue.
- CK II-mediated phosphorylation of fragmin does not impact its interaction with actin.
- The independent phosphorylation of both fragmin and actin suggests complex regulatory mechanisms within the actin cytoskeleton.