Article Figures & Data
Figures
- Figure 1.
Overview of the BTK locus in humans. (A) BTK-C transcription is initiated at an alternative transcriptional start site. Encoded sequence in this exon is phylogenetically conserved in mammals. (B) The key feature encoded in this sequence is one or more predicted palmitoylation sites. (C) The BTK-C isoform is expressed in approximately 15% of breast cancer tumors. BTK-C expression is not correlated with sample type. (D) Volcano plot of reverse-phase protein array data of BTK-C -expressing and -non-expressing tumors from the TCGA Firehose Legacy dataset. Only those protein species which have statistically significant (p<0.05 and q<0.05) differences in abundance between the two groups are labeled. Human breast tumors that do not express BTK-C are enriched in common breast cancer signaling inputs including ERBB2, the estrogen receptor, androgen receptor and the PIP3-dependent serine threonine kinase, PDPK1.
- Figure 2.
The BTK-C isoform is palmitoylated. (A) Diagram of BTK isoform C-terminal Flag-tagged constructs. (B) Expression of Flag-tagged constructs in MDA-MB-231 cells. (C) Detection scheme for acylated BTK isoforms. Cells were incubated with 17-ODYA. Flag immunoprecipitates were reacted with azide-activated biotinylation reagent and detected through reaction with streptavidin-linked horseradish peroxidase. (D) HEK293 cells were transfected with 1μg of BTK-A, BTK-C, or BTK-C C13A,C16A and 2 μg of a plasmid expressing zDHHC5. 24h after transfection cells were incubated for 4 h with 100 μM palmitic acid alkyne (17-ODYA). Cell lysate supernatants were immunoprecipitated with Anti-Flag M2 affinity gel (Sigma) at 4°C overnight, subjected to click chemistry with 20 μM biotin picolyl azide for 30 m at room temperature before electrophoresis and blotting.
- Figure 3.
Subcellular distribution of the BTK isoforms in breast cancer cells. (A) Diagram of BTK pleckstrin homology domain with GFP reporters. Expression of each reporter was driven by a CMV promoter in a retroviral vector. (B) Localization of wild type and mutant isoform reporters in breast cancer cell lines MD-AMB-231 (triple negative; PTEN+), BT549 (triple negative; PTEN-), SUM149 (triple negative; PTEN-) and SKBr3 (luminal HER2 enriched; PTEN+). BTK-A isoform is found in the plasma membrane (red arrows) and to a greater degree in the nuclei of cancer cells (white arrows). BTK-C is also found on the plasma membrane but exhibits a greater degree of perinuclear localization (gray arrows). Mutation of the two palmitoylation sites reduces membrane localization of BTK-C. Mutation of arginine 28 of BTK (arginine 62 and BTK-C sequence) also decreases membrane staining. (C) Subcellular fractionation showing increased levels of BTK-A in the nucleus and greater BTK-C in membrane fractions. (D) BTK reporters localize with actin in cancer cells. Scale bars: 100 μm.
- Figure 4.
BTK isoform localization and activation is responsive to PI3K pathway signaling in solid tumor cells. (A) BTK isoform localization in MDA-MB-231 (PTEN+) and SUM149 (PTEN-) cells. (B) Pharmacological inhibition of PI3K decreases association of BTK-A and BTK-C with the plasma membrane in LNCaP C4-2b (PTEN-) cells. Scale bar: 100 μm. (C) Activating tyrosine phosphorylation of the BTK isoforms in transiently transfected HEK293 cells. Y551 phosphorylation is dependent on other kinases, Y223 autophosphorylation results from BTK activation. Values represent fold tyrosine-phosphorylated signal normalized to total transfected BTK control (anti-Flag) determined by densitometry. (D) PIP3 dependence of BTK-C activation in transfected LNCaP C4-2b (PTEN-) cells. PTEN activity by expression of PTEN dominant negative mutants increases BTK-C activation. Additionally, a BTK-C non-kinase domain construct (PH-EGFP) capable of dimerizing with full length BTK reduces activating phosphorylation. In each case, 24 hours after transfection cell lysates were collected for immunoblotting.
- Figure 5.
(A) Activating tyrosine phosphorylation of the BTK isoforms. BTK-A and BTK-C constructs were transfected into HEK293 cell and after 24 hours stimulated with insulin. Values represent fold tyrosine-phosphorylated signal normalized to total BTK-A or BTK-C (anti-Flag) determined by densitometry. (B) Phosphorylation of the BTK target tyrosine 759 of PLCγ2 in transfected HEK293 cells. (C) BTK-C expression increases proliferation rate of MDA-MB-231 (PTEN+) but not SUM149 (PTEN-) cells. Cells were seeded in 96 well plates and fixed with 4% formaldehyde and counted at 72 hours. Data were normalized to control and are presented as mean±SD. *p<0.05 using Student’s t-test, n=3. (D) BTK-C increases glucose uptake in MDA-MB-231 (PTEN+) but not SUM149 (PTEN-) cells. Cells were seeded and treated with 100 μM 2-NBDG for 15 min. Fluorescence images were acquired with an InCell 2200 and overall fluorescence intensity quantified. Data were normalized to control and are presented as mean±SD. *p<0.05 using Student’s t-test, n=3.
- Figure 6.
Alternative N-terminal kinases in the human genome. (A) Exon maps of kinases with alternative first exons that either do or do not contain palmitoylation sites, as occurs with BTK-A and BTK-C. (B) Alternate N terminal sequences of tyrosine kinases TXK, FRK, HCK, ABL1, ABL2, and the serine/threonine kinase PDPK1 resemble BTK. Isoforms containing palmitoylation sequences in the amino terminal region are enriched in cysteine and methionine residues in predicted protein products. The encoded alternative isoforms represent a binary switch as non-palmitoylated forms are depleted of cysteine and non-start codon methionine residues near the N terminus.
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