Isc10 is a Dual Mechanism Inhibitor of CMGC Kinases

Abstract

Autophosphorylation of the activation loop is a common step that converts many protein kinases from an inactive to an active state. Despite the physiological importance of these reactions, the mechanisms that control activation loop autophosphorylation are poorly understood. Smk1 is a meiosis‐specific MAPK in yeast that autophosphorylates its activation loop on tyrosine (Y), thereby upregulating its own catalytic activity. Previously, we identified Isc10 as an inhibitor of Smk1. Here, we show that Isc10 inhibits Smk1 by two mechanisms. First, the C‐terminal segment of Isc10 binds to Smk1 and specifically inhibits the cis‐autophosphorylation of Y209 in its activation‐loop. Second, prior to Smk1 autophosphorylation, the low activity state Smk1 that is bound to Isc10 phosphorylates it on serine 97 (S97). The phosphorylated S97, in turn, becomes trapped in the Smk1 active site. This prevents phosphorylation of downstream targets before activating signals promote Isc10 degradation and the release of active Smk1. Further, Isc10 inhibits autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. Taken together, these findings define a novel class of developmentally‐regulated molecules that inhibit CMGC‐group kinases.

The MAPK homolog, Smk1, promotes assembly of the glucan layer of the spore wall in S. cerevisiae

“…To further examine the role of Smk1 in regulating the spore wall, we used an analog-sensitive form of Smk1 in which the "gatekeeper" glutamine near the ATP-binding pocket (Q121) had been changed to alanine (smk1-as1) (Rimal et al, 2022). While smk1-as1 diploids form spores that are morphologically indistinguishable from wild-type spores, the bulky purine analog inhibitor, PP1, sharply reduced spore formation in the smk1-as1 background (but not in the wild-type) (Rimal et al, 2022). The formation of spores was also specifically sensitive to PP1 in the cda1,2Δ smk1-as1 background.…”

“…The resulting KanR::smk1-as1 fragment was then transformed into a haploid derived from the SMK1-His8-HA::LEU2 strain LRY253 (Whinston et al, 2013) to generate the KanR::smk1-as1-His8-HA::LEU2 strain JTY1063 that was outcrossed to generate strains of opposite mating types that were mated to produce the homozygous KanR::smk1-as1-His8-HA diploid. To inhibit Smk1-as1, the purine analog PP1 was added from a 10 mmol stock solution in DMSO to a final concentration of 10 µmol as previously described (Rimal et al, 2022).…”