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Isc10, an inhibitor of the Smk1 MAPK, prevents activation loop autophosphorylation and substrate phosphorylation
Abhimannyu Rimal1, Thomas M Swayne1, Zeal P Kamdar1
1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Abstract:
Many eukaryotic protein kinases are activated by the intramolecular autophosphorylation of activation loop residues. Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that autophosphorylates its activation loop tyrosine and thereby upregulates catalytic output. This reaction is controlled by an inhibitor, Isc10, that binds the MAPK during meiosis I and an activator, Ssp2, that binds Smk1/Isc10 during meiosis II. Upon completion of the meiotic divisions, Isc10 is degraded, and Smk1 undergoes autophosphorylation to generate the high activity form of the MAPK that controls spore formation. How Isc10 inhibits Smk1 is not clear. Here, we use a bacterial coexpression/reconstitution system to define a domain in the carboxy-terminal half of Isc10 that specifically inhibits Smk1 autophosphorylation. Nevertheless, Smk1 bound by this domain is able to phosphorylate other substrates, and it phosphorylates the amino-terminal half of Isc10 on serine 97. In turn, the phosphorylated motif in Isc10 inhibits the Smk1 active site. These data show that Isc10 inhibits autophosphorylation and the phosphorylation of substrates by separate mechanisms. Furthermore, we demonstrate Isc10 can inhibit the autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. These findings define a novel class of developmentally regulated molecules that prevent the self-activation of MAPKs and MAPK-like enzymes.
Insights
A novel inhibitor, Isc10, controls yeast Smk1 (mitogen-activated protein kinase) activity by blocking its self-phosphorylation and substrate phosphorylation via distinct mechanisms, suggesting conserved regulation in eukaryotes.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Eukaryotic protein kinases, including yeast Smk1 (a meiosis-specific MAPK), are often activated by autophosphorylation of their activation loop.
- Smk1 activity is regulated by inhibitor Isc10 during meiosis I and activator Ssp2 during meiosis II.
- Isc10 degradation and Smk1 autophosphorylation are required for spore formation post-meiosis.
Purpose of the Study:
- To elucidate the mechanism by which Isc10 inhibits Smk1 activity.
- To identify the specific domain of Isc10 responsible for Smk1 inhibition.
- To investigate whether the inhibitory mechanism is conserved in mammalian kinases.
Main Methods:
- Bacterial coexpression and in vitro reconstitution system to study Smk1-Isc10 interactions.
- Site-directed mutagenesis to define the inhibitory domain of Isc10.
- In vitro kinase assays to assess Smk1 autophosphorylation and substrate phosphorylation.
- Testing Isc10 inhibition on mammalian intestinal cell kinase (ICK1/CILK1).
Main Results:
- A specific inhibitory domain within the carboxy-terminal half of Isc10 was identified.
- Isc10 inhibits Smk1 autophosphorylation via one mechanism and substrate phosphorylation via a separate mechanism.
- Smk1 phosphorylates Isc10 at serine 97, and this phosphorylation event inhibits the Smk1 active site.
- Isc10 also inhibits the autophosphorylation of the mammalian kinase ICK1 (CILK1).
Conclusions:
- Isc10 employs distinct mechanisms to inhibit Smk1 autophosphorylation and substrate phosphorylation.
- Phosphorylation of Isc10 at Ser97 is crucial for its inhibitory function.
- The findings reveal a conserved mechanism for regulating MAPK-like enzymes, identifying a novel class of developmental regulators.
- This study provides insights into the precise molecular control of meiosis-specific MAPK signaling.
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