Environmental Suppression of Neurospora crassa cot-1 Hyperbranching: a Link between COT1 Kinase and Stress Sensing

Abstract

cot-1 mutants belong to a class of Neurospora crassa colonial temperature-sensitive (cot) mutants that exhibit abnormal polar extension and branching patterns when grown at restrictive temperatures. cot-1 encodes a Ser/Thr protein kinase that is structurally related to the human myotonic dystrophy kinase which, when impaired, confers a disease that involves changes in cytoarchitecture and ion homeostasis. When grown under restrictive conditions, cot-1 cultures exhibited enhanced medium acidification rates, increased relative abundance of sodium, and increased intracellular glycerol content, indicating an ion homeostasis defect in a hyperbranching mutant. The application of ion transport blockers led to only mild suppression of the cot-1 phenotype. The presence of increased medium NaCl or sorbitol, H 2 O 2 , or ethanol levels significantly suppressed the cot-1 phenotype, restored ion homeostasis, and was accompanied by reduced levels of cyclic AMP-dependent protein kinase (PKA) activity. The cot-1 phenotype could also be partially suppressed by direct inhibition of PKA with KT-5720. A reduced availability of fermentable carbon sources also had a suppressive effect on the cot-1 phenotype. In contrast to the effect of extragenic ropy suppressors of cot-1, environmental stress-related suppression of cot-1 did not change COT1 polypeptide expression patterns in the mutant. We suggest that COT1 function is linked to environmental stress response signaling and that altering PKA activity bypasses the requirement for fully functional COT1.

The Nuclear Dbf2-Related Kinase COT1 and the Mitogen-Activated Protein Kinases MAK1 and MAK2 Genetically Interact to Regulate Filamentous Growth, Hyphal Fusion and Sexual Development in Neurospora crassa

“…This is best characterized by the gulliver-type suppression of the cot-1(ts) growth defects at restrictive conditions observed in mutants that harbor mak-2 pathway deletions. We have recently presented evidence indicating that inhibiting PKA activity can suppress the cot-1(ts) phenotype (Gorovits and Yarden 2003;Seiler et al 2006). Here, we demonstrate that the loss of MAK2 activity can also partially suppress the cot-1(ts) phenotype.…”

“…Deletion of mak2 is accompanied by a reduction in PKA activity: The suppression of cot-1(ts) by Dmak-2 resembled the previously described environmental suppression of cot-1(ts) and pod-6(ts) by external stresses (Gorovits and Yarden 2003;Seiler et al 2006). As environmental suppression of both kinases was correlated with reduced PKA activity levels, we analyzed PKA activity in the Dmak-2 strain and found several lines of evidence for reduced PKA activity.…”

“…Protein extraction, immunoblotting, and PKA activity measurement: Western blot analysis was performed as previously described (Gorovits and Yarden 2003). Briefly, N. crassa mycelial samples were frozen in liquid nitrogen, pulverized, and suspended in lysis buffer [1 m sorbitol, 10 mm HEPES (pH 7.5), 5 mm EDTA, 5 mm EGTA, 5 mm NaF, 0.1 m KCl, 0.2% Triton X-100, and complete protease inhibitor mixture (Roche Applied Science)].…”

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

“…The cot-1 gene encodes a Ser/ Thr protein kinase (Yarden et al 1992), and the colonial temperature-sensitive ( cot ) mutants exhibit abnormal polar extension and branching patterns when grown at the restrictive temperature. The cot-1 phenotype could be partially suppressed by direct inhibition of PKA with KT-5720 (Gorovits and Yarden 2003).…”

Regulation of polarised growth in fungi

“…cot-1 -mutants are suppressed by culture and stress conditions that lower cAMP levels and by an inhibitor, KT5720, that inhibits cAMP-dependent protein kinase (PKA) (Gorovits and Yarden, 2003).…”