Related Experiment Videos
SETA/CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and
Mirko H H Schmidt1, Baihua Chen, Lisa M Randazzo
1William and Karen Davidson Laboratory of Brain Tumor Biology, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, 2799 West Grand Blvd, Detroit, MI 48202, USA.
Abstract:
The adaptor protein SETA/CIN85/Ruk is involved in regulating diverse signal transduction pathways, including the internalization of tyrosine kinase receptors via the Cbl ubiquitin ligases, and attenuating PI3K activity by interaction with its regulatory subunit. Here we present evidence for a new aspect of SETA function, based on the initial observation that it co-localizes with actin in microfilaments and at focal adhesions, and with microtubules. Although there was no evidence for direct molecular interactions between SETA and cytoskeletal proteins, the SETA-interacting protein AIP1, which is a rat ortholog of the Xenopus src substrate Xp95, strongly interacted with structural proteins of the cytoskeleton, including actin and tubulins. Both SETA and AIP1 interacted with focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (PYK-2), and c-Cbl interacted with PYK-2. AIP1, which interacted more strongly than either SETA or c-Cbl, required an intact consensus tyrosine kinase phosphorylation sequence at Y319 to bind to focal adhesion kinases, which suggests that phosphorylation is an important mediator of this complex. SETA, which interacted as a dimer with focal adhesion kinases, promoted the interaction between PYK-2 and AIP1. Direct analysis of the impact of these proteins on cell adhesion, by use of an electrical cell-substrate impedance sensor (ECIS), showed that SETA promoted cell adhesion while AIP1 and c-Cbl reduced it. Furthermore, the ability of AIP1 and AIP1 mutants to decrease cell adhesion in ECIS analysis correlated with their presence in PYK-2 complexes, providing a direct link between AIP1-mediated molecular interactions and cellular behavior. Transfection of AIP1 also reduced the level of phosphorylation of endogenous PYK-2 and FAK, suggesting that this protein may directly regulate focal adhesion kinases, and thereby cell adhesion. These data are the first to implicate the adaptor protein SETA and its binding partner AIP1 as being involved with the cytoskeleton and in the regulation of cell adhesion, and suggest that they may be part of the focal adhesion kinase regulatory complex.
Insights
The adaptor protein SETA and its partner AIP1 interact with cytoskeletal proteins and focal adhesion kinases, influencing cell adhesion. SETA promotes cell adhesion, while AIP1 and c-Cbl reduce it, linking these proteins to cytoskeleton regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The adaptor protein SETA/CIN85/Ruk regulates tyrosine kinase receptor internalization and PI3K activity.
- SETA has been observed to co-localize with cytoskeletal components like actin and microtubules.
Purpose of the Study:
- To investigate a novel function of SETA involving the cytoskeleton and cell adhesion.
- To elucidate the role of SETA-interacting protein AIP1 in these processes.
Main Methods:
- Co-localization studies of SETA and AIP1 with cytoskeletal proteins.
- Co-immunoprecipitation assays to identify protein interactions (SETA, AIP1, FAK, PYK-2, c-Cbl).
- Electrical Cell-Substrate Impedance Sensing (ECIS) to measure cell adhesion.
- Analysis of PYK-2 and FAK phosphorylation levels.
Main Results:
- SETA co-localizes with actin and microtubules; AIP1 strongly interacts with actin and tubulins.
- SETA, AIP1, and c-Cbl interact with focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2).
- SETA promotes cell adhesion, while AIP1 and c-Cbl reduce it, correlating AIP1's effect with PYK-2 complex presence and reduced PYK-2/FAK phosphorylation.
Conclusions:
- SETA and its binding partner AIP1 are implicated in cytoskeleton regulation and cell adhesion.
- These proteins may form part of the focal adhesion kinase regulatory complex, influencing cell behavior.
- AIP1's interaction with PYK-2, mediated by phosphorylation, is crucial for regulating cell adhesion.