Integrated signaling and transcriptome analysis reveals Src family kinase individualities and novel pathways controlled by their constitutive activity

The Src family kinases (SFKs) Lck and Lyn are crucial for lymphocyte development and function. Albeit tissue-restricted expression patterns the two kinases share common functions; the most pronounced one being the phosphorylation of ITAM motifs in the cytoplasmic tails of antigenic receptors. Lck is predominantly expressed in T lymphocytes; however, it can be ectopically found in B-1 cell subsets and numerous pathologies including acute and chronic B-cell leukemias. The exact impact of Lck on the B-cell signaling apparatus remains enigmatic and is followed by the long-lasting question of mechanisms granting selectivity among SFK members. In this work we sought to investigate the mechanistic basis of ectopic Lck function in B-cells and compare it to events elicited by the predominant B-cell SFK, Lyn. Our results reveal substrate promiscuity displayed by the two SFKs, which however, is buffered by their differential susceptibility toward regulatory mechanisms, revealing a so far unappreciated aspect of SFK member-specific fine-tuning. Furthermore, we show that Lck- and Lyn-generated signals suffice to induce transcriptome alterations, reminiscent of B-cell activation, in the absence of receptor/co-receptor engagement. Finally, our analyses revealed a yet unrecognized role of SFKs in tipping the balance of cellular stress responses, by promoting the onset of ER-phagy, an as yet completely uncharacterized process in B lymphocytes.

Incubation with Dox revealed a time-dependent induction of Lck expression with highest levels being achieve after 48h (data not shown), thus all subsequent analyses were performed 48h after Dox addition.Non-Dox treated cultures invariably displayed baseline staining with all antibodies used in this study, and regardless of the constructs they had been transduced with (data not shown).Therefore, for all subsequent analyses, -Dox samples are a mixture of cell lines used for each corresponding experiment.

Fig S1. Development of a B cell model system for ectopic Lck expression A. Comparison of Lck Dox-inducible expression in B cell lines
The indicated B-cell lines were stably transduced with the lentiviral vector pLVX-Tight-Puro encoding Lck WT. 48h after Dox addition, cells were stained for Lck total protein expression and analysed by FACS.JCaM1.6 cells (an Lck-deficient jurkat variant) were used as a negative control for a-Lck antibody staining.Bar graph indicated a-Lck MFIs (n=2).

B. The Raji cell line lacks surface BCR expression
Unfixed live Lck-transduced Raji and BJAB cells were stained for CD79b surface expression, Jurkat cells were used as negative control for a-CD79b antibody staining.

A. Ectopic Lck dramatically increases levels of SFK activity in BJAB cells
Lck-expressing BJAB cells and their non-Dox treated counterparts were stained for Lck and pY416 and analysed by FACS.MFI values for each antibody staining were determined by analysis with the Flowjo software.Graphs show increases in total protein expression and SFK activity levels (n=3, Unpaired Student t test; mean +/-SD; ***P < 0.001).

B. Ectopic Lck in BJAB does not affect the activation status of Lyn or other B-cell expressed SFKs
Left-hand side.Endogenous Lyn was immunoprecipitated (IP) from the Lck-BJAB cell line in the presence or absence of Dox.Lyn IPs and total cell lysates were analysed by western blotting with a-pY416 (upper panels) and a-Lyn (lower panels) antibodies.Actin blot (middle panel) verifies equal sample loading for the cell lysates.JCaM1.6 cells (devoid of both Lyn and Lck expression) were used as a negative control.Increased reactivity of the a-pY416 antibody in the +Dox sample of total Lysates (but not the IPs) is due to ectopic Lck (Fig S2A ).
Right-hand side.Total cell lysates from BJAB cells expressing GFP-tagged Lck or Lyn and their non-Dox treated counterparts were analysed by western blotting with a-pY416.The presence of GFP increases the MW of the kinases, thus allowing visualization of both the tagged proteins and endogenous levels of active SFKs (as indicated by the corresponding arrows).Actin blot (lower panel) verifies equal sample loading.

C. Ectopic Lck does not increase global tyrosine phosphorylation in HEK293T human embryonic kidney cells
Total cell lysates of HEK293T cells transiently transfected with empty vector (EV) or the indicated SFK members, were analysed by western blotting with the global a-phosphotyrosine antibody, clone 4G10.Lower-panels, a-pY416 and a-actin blots of the same samples verify equal levels of activity amongst individual SFKs and sample loading, respectively.

B. FACS cell sorting of GFP+ populations
GFP histograms of Lck-and Lyn-expressing cells prior (upper panels) and after (lower panel).Fluorescence activated cell isolation of GFP+ populations (as indicated by the corresponding gates on the upper panels histograms).
Sorted cells were cultured and used for all subsequent experimental analyses.

C. Lck-BJAB cells have higher levels of GFP expression and consequently SFK activity on a whole cell population basis
BJAB-Lck and BJAB-Lyn cells were stained with a-pY416 and analysed by FACS in parallel to their -Dox counterparts.2D FACS plots demonstrate the dose-response relationship between protein expression (GFP) and SFK activity (pY416).Graphs depict MFI values within the whole cell population for each cell line (n=13, Unpaired Student t test; mean +/-SD; **P < 0.01, ****P < 0.0001).

D. Equivalent levels of Lck and Lyn expression in transfected HEK293T cells
Histograms of HEK 293T cells were transiently transfected with the indicated constructs and analysed by FACS for GFP and a-pY416 fluorescence.The indicated gates for GFP+ and pY416+ cells were set on empty vector transfected cells.

E. Equal GFP gating strategy
Our studies required comparison of Lyn and Lck on an equivalent protein-expression basis.Since it was unattainable to obtain Lyn expression similarly high to Lck in B-cell lines, we resolved in following an "equal GFP gating strategy".In the illustrated example, FACS analysed samples of cells stained for pCD79a, are displayed as 2D FACS plots versus GFP fluorescence.Rectangle gating isolates populations of Lck-and Lyn-expressing cells with equal GFP MFIs (upper panels).These populations are further subdivided by quadrant gates set on the negative control (-Dox) sample (lower panels).The frequency of cells displayed in the upper right quadrant (double-positive for SFK expression and pCD79a) is used for further analysis.

Figure S4. Lck and Lyn provide a relative advantage in the magnitude of a-IgM triggered responses
A. BJAB-Lck or BJAB-Lyn cells were either left untreated or were stimulated with a-IgM for 10 min at 37 o C prior to staining with the indicated antibodies and analysed by FACS.Graphs depict frequency of cells double-positive for SFK expression and pCD79a, pSyk and pAkt for the whole cell population (n≥4, Unpaired Student t test; mean +/-SD; *P < 0.05, **P < 0.01; ns: not significant).
B. anti-phosphotyrosine (4G10) and actin western blots of total cell lysates of the indicated samples.Stimulation conditions as in A.

Figure
Figure S3.GFP-tagged SFK behavior and sorting A. Addition of C-terminal GFP tag in Lck and Lyn does not alter their activation and substrate phosphorylation capacity

Fig S5 .
Fig S5.Doxycycline does not affect representative gene expression in BJAB cells RT-qPCR for the validated RNA-seq derived differentially expressed genes shown in Fig. 3E, in parental BJAB cells cultured in the absence or presence of Dox.The y-axis shows the log2FC of the gene expression of the parental cell line +Dox compared to the same cell line -Dox (n=2, Multiple unpaired Student t test; mean +/-SD, no statistical significance).

Table S1 .
Specific primers for genes used in the qPCR.