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The FKBP12-rapamycin-associated protein (FRAP) is a CLIP-170 kinase
Jae H Choi1, Paula G Bertram, Ryan Drenan
1Myriad Proteonomics, Inc., Salt Lake City, UT 84116, USA.
Abstract:
CLIP-170/Restin belongs to a family of conserved microtubule (MT)-associated proteins, which are important for MT organization and functions. CLIP-170 is a phosphoprotein and phosphorylation is thought to regulate the binding of CLIP-170 to MTs. However, little is known about the kinase(s) involved. In this study, we show that FKBP12-rapamycin-associated protein (FRAP, also called mTOR/RAFT) interacts with CLIP-170. CLIP-170 is phosphorylated in vivo at multiple sites, including rapamycin-sensitive and -insensitive sites, and is phosphorylated by FRAP in vitro at the rapamycin-sensitive sites. In addition, rapamycin inhibited the ability of CLIP-170 to bind to MTs. Our observations suggest that multiple CLIP-170 kinases are involved in positive and negative control of CLIP-170, and FRAP is a CLIP-170 kinase positively regulating the MT-binding behavior of CLIP-170.
Insights
FKBP12-rapamycin-associated protein (FRAP) interacts with microtubule-associated protein CLIP-170, phosphorylating it at specific sites. This phosphorylation by FRAP positively regulates CLIP-170
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule-associated proteins (MAPs) like CLIP-170 are crucial for microtubule organization and function.
- CLIP-170 is a phosphoprotein, and its phosphorylation status is believed to regulate its interaction with microtubules.
- The specific kinases responsible for CLIP-170 phosphorylation remain largely unidentified.
Purpose of the Study:
- To investigate the interaction between CLIP-170 and potential kinases.
- To identify the kinase(s) involved in CLIP-170 phosphorylation.
- To determine the functional consequence of CLIP-170 phosphorylation on its microtubule-binding activity.
Main Methods:
- Co-immunoprecipitation assays to detect protein interactions.
- In vitro kinase assays to assess phosphorylation activity.
- In vivo phosphorylation studies using rapamycin treatment.
- Microtubule binding assays.
Main Results:
- FKBP12-rapamycin-associated protein (FRAP, also known as mTOR/RAFT) was found to interact with CLIP-170.
- CLIP-170 undergoes phosphorylation at both rapamycin-sensitive and -insensitive sites in vivo.
- FRAP phosphorylates CLIP-170 in vitro at rapamycin-sensitive sites.
- Rapamycin treatment inhibited the microtubule-binding ability of CLIP-170.
Conclusions:
- Multiple kinases regulate CLIP-170 activity through phosphorylation.
- FRAP is identified as a kinase that positively regulates CLIP-170's interaction with microtubules.
- Phosphorylation by FRAP is a key mechanism controlling CLIP-170's function in microtubule dynamics.