Abstract
The two PKC orthologs Pck1 and Pck2 in the fission yeast Schizosaccharomyces pombe operate in a redundant fashion to control essential functions, including morphogenesis and cell wall biosynthesis, as well as the activity of the cell integrity pathway and its core element, the MAPK Pmk1. We show here that, despite the strong structural similarity and functional redundancy of these two enzymes, the mechanisms regulating their maturation, activation, and stabilization have a remarkably distinct biological impact on both kinases. We found that, in contrast to Pck2, putative in vivo phosphorylation of Pck1 within the conserved activation loop, turn, and hydrophobic motifs is essential for Pck1 stability and biological functions. Constitutive Pck activation promoted dephosphorylation and destabilization of Pck2, whereas it enhanced Pck1 levels to interfere with proper downstream signaling to the cell integrity pathway via Pck2. Importantly, although catalytic activity was essential for Pck1 function, Pck2 remained partially functional independent of its catalytic activity. Our findings suggest that early divergence from a common ancestor in fission yeast involved important changes in the mechanisms regulating catalytic activation and stability of PKC family members to allow for flexible and dynamic control of downstream functions, including MAPK signaling.
Highlights
The protein kinase C family of isozymes plays essential roles in signaling pathways controlling cell growth, proliferation, differentiation, and cell death (1, 2)
Novel, and atypical mammalian PKC isoforms share a basic structure with a variable N-terminal regulatory domain followed by a highly conserved C-terminal kinase domain, which contains three conserved phosphorylation sites critical for catalytic activity: the activation loop (AL),[3] turn motif (TM), and hydrophobic motif (HM) (1, 2)
The PKC orthologs Pck[1] and Pck[2] share an essential role to modulate cell growth and morphogenesis in fission yeast (6, 7). Taking into account their redundant functions and strong structural similarity in the regulatory and catalytic domains, it might be anticipated that the mechanisms responsible for catalytic activation and stabilization of both kinases should be identical
Summary
Pck[1] is phosphorylated in vivo by Ksg[1] within the AL at Thr[823] and is more stable than Pck[2]. The levels of total (anti-HA antibody) and Thr(P)[823] Pck[1] were approximately 10 times higher than in ksg[1–208] cells either growing at the permissive temperature (25 °C) or incubated at 36 °C, which is a restrictive temperature for Ksg[1] function (Fig. 2B) These results indicate that Ksg[1] is responsible for AL phosphorylation of Pck[1] at Thr[823] in vivo, and this event might regulate Pck[1] stability (see below). We tested the ability of genomic versions of wildtype or mutated alleles of Pck[1] to suppress several known phenotypes of pck1⌬ cells, including defective signaling to the CIP and growth sensitivity in the presence of caspofungin, Calcofluor white, and magnesium chloride (12, 16), as biological readouts to comparatively assess their function in vivo. The increase in basal Pmk[1] phosphorylation displayed by the rho[1–596] hypomorphic allele (24) and pck1-A399E cells (Fig. 5C) was significantly reduced in a rho[1–596] pck1-A399E background
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