ISC10, a Developmentally Regulated Inhibitor of Non-Canonical MAPK Signaling
Mitogen-activated protein kinases (MAPKs) are signaling enzymes that play critical roles in human health and disease. In typical MAPK pathways, kinase cascades activate dual specificity MAPK kinases that phosphorylate threonine (T) and tyrosine (Y) residues in the activation-loop of the MAPK. These well-studied pathways control cellular responses to a variety of molecules in the extracellular environment. In atypical MAPK pathways, the MAPK is activated by autophosphorylation of the activation-loop. These pathways often transduce intracellular signals and developmental cues. Although atypically activated MAPKs control essential biological processes, the mechanisms that regulate atypical MAPK signaling remain poorly understood. In this thesis, I investigate how intracellular signals control an atypical MAPK in the context of a developmental program. These studies have provided novel mechanistic insights that are broadly applicable to how atypical MAPKs and MAPK-like enzymes are regulated. SMK1 encodes an atypically activated MAPK in yeast that triggers key steps in gamete (spore) differentiation. In this developmentally-regulated pathway, Smk1 is produced as cells enter the first meiotic division (MI). At this time, the cyclin-dependent kinase (CDK) activating kinase, Cak1, phosphorylates Smk1 on its activation-loop threonine (T207). As cells enter the second meiotic division (MII), an activator protein, Ssp2 is produced that triggers the autophosphorylation of Smk1 on its activation loop tyrosine (Y209). Prior to the work presented in this thesis, it had been shown that the Anaphase Promoting Complex/Cyclostome (APC/C) E3 ubiquitin ligase, bound to the meiosis-specific co-activator, Ama1, is required for the Ssp2-dependent activation of Smk1. These findings suggested that the APC/CAma1, which also promotes exit from MII, links the completion of meiosis to Smk1 activation. I began this study by hypothesizing that APC/CAma1 targets an inhibitor of Smk1 for ubiquitin-dependent proteosomal degradation. This thesis describes the identification and validation of Isc10 as an inhibitor of Smk1 that is targeted for destruction by the APC/CAma1. These data support a stepwise model connecting the activation of Smk1 to the APC/CAma1 through Isc10 will be presented. In this model, Smk1/Isc10 complexes (inhibited; I-complexes) are assembled during MI. Ssp2 binds to I-complexes during MII to form Smk1/Ssp2/Isc10 complexes (poised; P-complexes) that are awaiting activation. I discovered that two separate segments of Isc10 inhibit Smk1. First, a conserved segment in the C-terminus of Isc10 termed YAI (for the Y [tyrosine] - Autophosphorylation Inhibitor) motif specifically inhibits Smk1 autophosphorylation on Y209. Although Smk1 in P-complexes has not yet autophosphorylated its activation-loop Y, it nevertheless exists in a low activity state that phosphorylates the bound Isc10 on serine 97 (S97). The pS97 motif of Isc10 inhibits the activity of monophosphorylated Smk1, likely by binding to the catalytic site of the MAPK. As cells exit MII, the APC/CAma1 ubiquitylates Isc10, thereby promoting the destruction of Isc10. This releases active Ssp2/Smk1 that undergoes autophosphorylation on Y209 to generate the dually phosphorylated high-activity form of the MAPK (active; A-complexes) that triggers spore differentiation. One of the earliest events that take place during the spore differentiation program is the assembly of multilayered spore walls. I found that Smk1 controls Gsc2, a 1,3-ß-glucan synthase required for assembly of the inner layer of the spore wall. These findings link the IPA model for Smk1 activation to one of the earliest events in spore differentiation and provide a mechanistic explanation of how the APC/C can link differentiation to the G0-like phase of the meiotic cell cycle. Further, I also discovered that Isc10 can prevent the autophosphorylation of the mammalian intestinal cell kinase, ICK, suggesting an evolutionarily conserved mechanism of action. Taken together, the experiments described in this thesis provide novel insights into mechanisms that regulate atypical MAPKs and how differentiation is coupled to the G0 phase of the cell cycle.
Biochemistry|Molecular biology|Cellular biology
Rimal, Abhimannyu, "ISC10, a Developmentally Regulated Inhibitor of Non-Canonical MAPK Signaling" (2023). ProQuest ETD Collection - Thomas Jefferson University. AAI30249633.