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Phosphorylation of the kinase suppressor of ras by associated kinases
D J Volle1, J A Fulton, O V Chaika
1Eppley Institute for Research in Cancer and Allied Diseases, Department of Biochemistry and Molecular Biology, Omaha, Nebraska 68198-6805, USA.
Abstract:
The kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. The physiological role of mammalian KSR is not known. We examined the mechanisms regulating the phosphorylation of this putative kinase in mammalian cells. Wild-type mouse KSR and a mutated KSR protein predicted to create a kinase-dead protein are phosphorylated identically in intact cells and in the immune complex. Phosphopeptide sequencing identified 10 in vivo phosphorylation sites in KSR, all of which reside in the 539 noncatalytic amino terminal amino acids. Expression of the amino terminal portion of KSR alone demonstrated that it was phosphorylated in the intact cell and in an immune complex in a manner indistinguishable from that of intact KSR. These data demonstrate that the kinase domain of KSR is irrelevant to its phosphorylation state and suggest that the phosphorylation of KSR and its association with a distinct set of kinases may affect intracellular signaling.
Insights
Mammalian kinase suppressor of Ras (KSR) phosphorylation is regulated by its amino-terminal region, not its kinase domain. This finding suggests KSR
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Protein phosphorylation
Background:
- Kinase suppressor of Ras (KSR) acts as a loss-of-function suppressor of activated Ras phenotypes in model organisms.
- The physiological role of mammalian KSR remains largely uncharacterized.
- Understanding KSR regulation is crucial for elucidating its function in mammalian intracellular signaling.
Purpose of the Study:
- To investigate the mechanisms governing the phosphorylation of mammalian KSR.
- To determine if the kinase activity of KSR is essential for its own phosphorylation.
- To identify the regions of KSR involved in its post-translational modification.
Main Methods:
- Utilized wild-type and kinase-dead mutant mouse KSR proteins.
- Employed cell-based phosphorylation assays and immune complex analysis.
- Performed phosphopeptide sequencing to identify in vivo phosphorylation sites.
- Expressed and analyzed the amino-terminal portion of KSR independently.
Main Results:
- Wild-type and kinase-dead KSR proteins exhibited identical phosphorylation patterns in intact cells and immune complexes.
- Ten in vivo phosphorylation sites were identified within the noncatalytic amino-terminal 539 amino acids of KSR.
- The amino-terminal region of KSR alone was phosphorylated similarly to the full-length protein.
- KSR's kinase domain was found to be irrelevant to its phosphorylation state.
Conclusions:
- Mammalian KSR phosphorylation is regulated independently of its catalytic kinase activity.
- The amino-terminal domain of KSR is the primary determinant of its phosphorylation.
- These findings suggest that KSR phosphorylation and its kinase associations may modulate intracellular signaling pathways.