Journal: Nature Communications

Article Title: Optical control of protein phosphatase function

doi: 10.1038/s41467-019-12260-z

Figure Lengend Snippet: Optical control of the MKP3 catalytic site. a Western blot analysis confirms expression of caged MKP3 only in the presence of NVC (1 mM). b Biochemical phosphatase assays show complete loss of activity for the caged enzyme, until 365 nm irradiation restores the wild-type enzyme. Error bars denote standard deviation from three biological replicates; ** p < 0.01 from unpaired two-tailed Student’s t -test. c Using an ERK2-mCherry reporter, the activity of wild-type, caged (at the catalytic cysteine), and photoactivated MKP3 was investigated in HEK293T cells. Following pathway stimulation, nuclear exclusion of an ERK2-mCherry reporter is observed for all MKP3 variants. Scale bar 10 μm; also see Supplementary Fig. . d Quantification of nuclear/cytoplasmic ratios shows successful nuclear translocation for the reporter only control; however, none of the MKP3 variants show any significant change. Error bars represent standard error of the mean from nine individual cells combined from three biological replicates. One-way ANOVA tests at the 60-minute time point results in p < 0.0001 for the reporter compared to all MKP3 samples, indicating this a significant difference. Comparing C293NVC –UV to C293NVC + UV results in p > 0.1, indicating no significant difference. Data are provided in the Source Data file

Article Snippet: To generate GST-ERK2 protein, Addgene plasmid pGEX-4T-1 3xFlag-ERK2 (#47573) was transformed into BL21(DE3) using LB broth supplemented with ampicillin (100 µg/mL) and protein expression was performed following a literature procedure at a 100 mL scale .

Techniques: Western Blot, Expressing, Activity Assay, Irradiation, Standard Deviation, Two Tailed Test, Translocation Assay

Journal: Nature Communications

Article Title: Optical control of protein phosphatase function

doi: 10.1038/s41467-019-12260-z

Figure Lengend Snippet: ERK activity in response to optical activation of the MKP3 catalytic site. a An ERK-KTR-mCherry reporter was utilized to report on the activity of endogenous ERK2 levels in the presence of wild-type, caged, and photoactivated MKP3 variants in HEK293T cells. Wild-type MKP3 prevents EGF-induced triggering of the reporter, while the caged MKP3 is only active following UV exposure (365 nm, 2 min). Scale bar 10 μm; also see Supplementary Fig. . b Quantification of the cytoplasmic/nuclear ratio for the MKP3 variants shows successful photoactivation of MKP3. Error bars represent standard error of the mean for nine individual cells combined from three biological replicates. One-way ANOVA tests at the 60-minute time point resulted in p < 0.0005 when comparing C293NVC –UV to C293NVC + UV, and reporter only to WT. No significant difference ( p > 0.1) was observed between WT and C293NVC + UV or reporter only and C293NVC –UV. Data are provided in the Source Data file

Article Snippet: To generate GST-ERK2 protein, Addgene plasmid pGEX-4T-1 3xFlag-ERK2 (#47573) was transformed into BL21(DE3) using LB broth supplemented with ampicillin (100 µg/mL) and protein expression was performed following a literature procedure at a 100 mL scale .

Techniques: Activity Assay, Activation Assay

Journal: Nature Communications

Article Title: Optical control of protein phosphatase function

doi: 10.1038/s41467-019-12260-z

Figure Lengend Snippet: Design strategy for optical control of the phosphatase-kinase interaction motif (KIM). a The crystal structure of ERK2 (tan) bound to the KIM peptide of MKP3 (gray) shows essential electrostatic interactions of R64 and R65 with D316 and D319 (PDB: 2FYS). b Irradiation of HCK removes the coumarin caging group (green) and restores a native lysine residue. c Replacement of R65 with HCK generates steric hindrance and prevents electrostatic interaction until light exposure release K65, which enables the ERK2-MKP3 interaction

Article Snippet: To generate GST-ERK2 protein, Addgene plasmid pGEX-4T-1 3xFlag-ERK2 (#47573) was transformed into BL21(DE3) using LB broth supplemented with ampicillin (100 µg/mL) and protein expression was performed following a literature procedure at a 100 mL scale .

Techniques: Irradiation