The N-terminal region of the Neurospora NDR kinase COT1 regulates morphology via its interactions with MOB2A/B

Abstract

Nuclear Dbf2p-related (NDR) protein kinases are important for cell differentiation and polar morphogenesis in various organisms, yet some of their functions are still elusive. Dysfunction of the Neurospora crassa NDR kinase COT1 leads to cessation of tip extension and hyperbranching. NDR kinases require the physical interaction between the kinase's N-terminal region (NTR) and the MPS1-binding (MOB) proteins for their activity and functions. To study the interactions between COT1 and MOB2 proteins, we mutated several conserved residues and a novel phosphorylation site within the COT1 NTR. The phenotypes of these mutants suggest that the NTR is required for COT1 functions in regulating hyphal elongation and branching, asexual conidiation and germination. Interestingly, while both MOB2A and MOB2B promote proper hyphal growth, they have distinct COT1-dependent roles in regulation of macroconidiation. Immunoprecipitation experiments indicate physical association of COT1 with both MOB2A and MOB2B, simultaneously. Furthermore, the binding of the two MOB2 proteins to COT1 is mediated by different residues at the COT1 NTR, suggesting a hetero-trimer is formed. Thus, although MOB2A/B may have some overlapping functions in regulating hyphal tip extension, their function is not redundant and they are both required for proper fungal development.

Fig. 1

Conserved residues within the N-terminal region of COT1 affect fungal morphology. A. Schematic summary of amino acid alterations introduced into COT1 NTR. The two highly conserved phosphorylation sites at the kinase domain (Ser417) and C Terminal domain (Thr589) are also shown. Grey box represents the NTR and a dotted box represents the kinase domain. The residues’ numbers refer to the COT1 protein (long transcript) without the Myc tag. B. Growth of myc-cot-1 and the NTR mutants harbouring the different Myc-tagged alleles of cot-1. Cultures were grown overnight at 34°C. C. Hyphal morphology at the edge of the colony of wild-type, cot-1(ts), myc-cot-1 and the NTR point-mutation strains cultured overnight at 34°C. Bar = 100 μm. D. Microscopic images of myc-cot-1, cot-1(R167A) and cot-1(R203A) hyphal tips grown at 34°C. Bar represents 10 μm.

Fig. 2

Morphological consequences of mutations within the COT1 NTR in a Δmob-2a and Δmob-2b background: Growth rate (A), branching (B) and conidiation (C) of myc-cot-1, Δmob-2a, Δmob-2b and mutants harbouring the different alleles carrying the MYC-tagged point mutations in cot-1 NTR. All experiments were conducted at 34°C, in triplicate. Black bars indicate myc-cot-1 background, white bars indicate a Δmob-2a background and grey bars indicate a Δmob-2b background. Error bars represent standard error of the mean.

Fig. 3

COT1 is expressed as three different isoforms: (A) Western-blot analysis of total cell extracts probed with anti-Myc antibodies. (B) Western blot analysis of IP with anti-myc detected with anti-myc antibodies. Arrows on the right indicate the three COT1 isoforms, with estimated size.

Fig. 4

Mutating COT1 Arg203 to Ala impairs COT1-MOB2A interactions: Growth curves of yeast harbouring the indicated two-hybrid plasmids in SD-Leu/-Trp liquid medium (black) or in SD high selection liquid medium -Leu/-Trp/-His/-Ade (white). Similar growth of the yeast strains in the two different media indicates strong physical interaction between the two proteins.

Fig. 5

Protein-protein interactions between the different alleles of COT1 NTR and the MOB2A/MOB2B proteins as determined by yeast two hybrid experiments. Different yeast transformants carrying MOB2A (A) or MOB2B (B) as a pray and the different COT1 alleles as a bait were grown overnight in SD medium lacking Leu and Trp. The supernatant of the yeast culture was assayed for α-galactosidase activity. High activity of α-galactosidase indicates strong interaction between the tested proteins. The results are an average of three different experiments that were performed in triplicate. Bar indicates standard error.

Fig. 6

Substitution of Glu198 with Ala abolishes MYC-COT1-MOB2B physical association. Silver stain detection of MYC-immunoprecipitated proteins of myc-cot-1, Δmob-2a, Δmob-2b, cot-1(E198A) and cot-1(E198A);Δmob-2a, resolved in a polyacrylamide gel.

Fig. 7

Mutations at the COT1 NTR affect COT1 dimerization as determined by Yeast Two Hybrid assay. Yeast transformants carrying the each of COT1 alleles, both as bait and as pray, were grown overnight in SD medium lacking Leu and Trp. The supernatant of the yeast culture was assayed for α-galactosidase activity. High α-galactosidase activity indicates strong interaction between the tested proteins. The results are an average of three independent experiments that were performed in triplicate. Bar indicates standard error.