Table 2.
In vitro kinase activities, autophosphorylation patterns, and relative growth rates of phosphosite-modified COT1 variants
| N. crassa abbreviated genotype | Abbreviated genotype Mean (±SD) COT1 activitya (n ≥ 3) | Ser417 autophosphorylation of COT1 |
COT1-MOB2 interaction | % relative growth rate (±SD) at 37°C (n ≥ 5) | |
|---|---|---|---|---|---|
| In vitro | In vivo | ||||
| myc-cot-1(T589E) | 100 | + | + | + | 65 ± 2 |
| myc-cot-1 (S417A,T589E) | 5–50b | − | − | + | 1 ± 1 |
| myc-cot-1(T589A) | 10 ± 4 | + | + | + | 1 ± 1 |
| myc-cot-1 | 5 ± 2 | + | + | + | 100 |
| Δpod-6; myc-cot-1 | 4 ± 1 | + | + | + | 0c |
| myc-cot-1(S417E) | 1 ± 0.5 | − | − | + | 76 ± 2 |
| myc-cot-1(S417A) | 0.04 ± 0.5 | − | − | + | 19 ± 2 |
| myc-cot-1; Δmob-2a; Δmob-2b | 0.04 ± 0.5 | − | +d | − | 0c |
| myc-cot-1; cot-1-tev-gfp | 0e | −e | −e | −e | 100 |
| myc-cot-1(D337A) | 0 | − | − | + | 0c |
a
Peptide-based kinase activities were determined using myc-COT1 as the reference and then recalculated relative to the myc-COT1(T589E) strain.
b
This COT1 variant produced highly variable kinase activities in several independent experiments, ranging from 5 to 50% of that of the myc-COT1(T589E) strain.
c
The indicated strains displayed phenotypes identical to those of the Δcot-1 and Δpod-6 strains. The growth rate of both deletion strains was ∼1 mm/week and is reported as 0.
d
Only the large isoform of COT1 was expressed and phosphorylated in a mob-2 double deletion background.
e
Characteristic of the COT1-COT1 dimer.