Figure 1.
Y416 is phosphorylated in closed, repressed c-Src.
AD293 cells were transfected with the indicated constructs and after 24h analysed by Western Blots as shown. Key: Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; β-gal, β -galactosidase in same vector as c-Src constructs A. The blot was standardized to c-Src levels. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. The blot was standardized to endogenous STAT3. Blots were performed twice and showed consistent results.
Figure 2.
Phosphorylation of Y416 in the closed, repressed conformation is driven by core kinase domains.
A. Confocal micrographs of c-Src expressed as fusions to Emerald in AD293 cells in either full length or truncated form lacking the first 63 residues (Δ1–63), which contains the unique domain and myristoylation sequences. Cells were co-transfected with mKate2-F, which is a fluorescent protein containing a farnesylation signal to localize it to membranes. Turquoise shows c-Src-Em, red shows mKate2-F. Scale bar, 10 µm. Images are representative of three independent experiments. B. Western Blots of AD293 cells transfected with the indicated constructs for 24 h with 10 µg total protein lysate. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. Key: Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; β-gal, β -galactosidase in same vector as c-Src constructs. Experiments were performed at least twice, which showed consistent results.
Figure 3.
Phosphorylation of Y416 requires kinase activity and is sustained upon suppression of activity.
AD293 cells were transfected with the indicated constructs and analysed by Western Blot 24 h post-transfection. Blots were performed at least twice, which showed consistent results; a single representative is shown. Key: KD, kinase dead K293 M mutant; Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; β-gal, β -galactosidase in same vector as c-Src constructs. A. Western Blot standardized for c-Src levels. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. Western Blot standardized for STAT3 levels.
Figure 4.
Expression-level dependence of c-Src on Y416 phosphorylation.
A. Western Blots of AD293 cells transfected with c-Src variants for 24 h standardized to β-tubulin. C-Src was transfected at different doses by adjusting the proportion of DNA used in transfections from 100% to 1.56% supplemented with a vector expressing non-fluorescent Y66L GFP derivative. Key: closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. Densitometry analysis of the Western Blot data in panel A for STAT3 phospho-Y705 immunoreactivity, c-Src immunoreactivity and c-Src phospho-Y416 immunoreactivity with antibody #1 (cat. PK1109). Blots shown are representative of three independent experiments. Densitometry analysis is for the blots shown.
Figure 5.
Open c-Src forms a minor proportion of high mass complex in cell lysate.
Sedimentation velocity data at low speed (3,000 rpm) by analytical ultracentrifugation of cell lysates of AD293 cells transfected with c-Src-Emerald is shown as raw data (gray incrementing lines), with the first scan shown in red and the last scan in blue. Data are representative of three independent experiments. Cells were transfected with two different doses of c-Src: either 100% c-Src in transfection, or 12.5% c-Src with the remainder of the DNA supplemented with a non-fluorescent Y66L derivative of Emerald. Under these conditions low mass forms of c-Src-Emerald such as monomers and dimers will not sediment. Brackets indicate the loss of fluorescence in the supernatant due to pelleting of high mass complex (>∼1000 S).
Figure 6.
Open and closed c-Src predominately form low mass oligomers in cell lysate.
Sedimentation velocity data at high speed (40,000 rpm) by analytical ultracentrifugation of cell lysates of AD293 cells transfected with c-Src-Emerald is shown as raw data (gray incrementing lines), with the first scan shown in red and the last scan in blue. Data shown are representative of three independent experiments. Fits to a c(s) model are shown in magenta dashed lines. Cells were transfected with two different doses of c-Src: either 100% c-Src in transfection, or 12.5% c-Src with the remainder of the DNA supplemented with a non-fluorescent Y66L derivative of Emerald.
Figure 7.
Open and closed c-Src form predominately monomers and dimers in cell lysate.
Size distributions of the c(s) fit to the data in Figure 6, as well as a control of Emerald-transfected cell lysate, are shown. Corresponding masses based on c(M) size distributions are shown with the arrow for the two major species. Three independent experiments produced consistent results; data shown are for a single experiment.
Figure 8.
Open c-Src forms high mass complexes and dimers to a greater extent than the closed and wild type forms.
A. Percent of c-Src that forms high mass complexes (>∼1000 S). Values were calculated as percent of fluorescence intensity lost from the supernatant at 6.8 cm radius between the first and last sedimentation velocity scans at low speed (3000 rpm). Loss of intensity is indicated by brackets in Figure 5. (Data shows mean ± S.D., n = 3). There was a significant difference between open and closed (P = <0.001), wild-type versus open (P = 0.001) but not wild-type versus closed (P = 0.362) as assessed by two-way ANOVA and Holm-Sidak pairwise comparison. There was no difference between 100% and 12.5% transfection conditions (P = 0.206; two-way ANOVA). B. Percent of low mass c-Src that exists in dimers. Values are calculated as the percent of material greater than 5.2 S from size distribution of c(s) fits to high speed (40,000 rpm) sedimentation velocity data. (Data shows mean ± S.D., n = 3). There was a significant difference between open and closed (P = 0.007) but not wild-type versus closed (P = 0.183) or wild-type versus open (P = 0.059) as assessed by two-way ANOVA and Holm-Sidak pairwise comparison. There was no difference between 100% and 12.5% transfection conditions (P = 0.760; two-way ANOVA).