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Blood, Vol. 110, Issue 7, 2511-2519, October 1, 2007
The Src family kinase Hck regulates mast cell activation by suppressing an inhibitory Src family kinase Lyn
Blood Hong et al.
110: 2511
Supplemental materials for Hong et al, Vol. 110, Issue 7, 2511-2519
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 77.6 KB)
- Figure S1. Estimation of cellular concentrations of three SFKs in mouse BMMCs (JPG, 49.7 KB)
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(A) Schematic of mouse Hck proteins composed of N-terminal unique, SH3, SH2, SH1 (kinase) and C-terminal regulatory domains. The p59 isoform has an extra N-terminal 21 amino acid sequence generated by alternative initiation of translation. Numbering of amino acid residues follows Holtzman et al.1 (B) Hck proteins are expressed in wt but not hck−/− spleens. Spleens from two individual mice of each genotype were examined for p59hck and p56hck expression. (C) Total cell lysates of wt or lyn−/− BMMCs were analyzed by immunoblotting. To quantify Lyn, Fyn, and Hck, predetermined amounts of GST fusion proteins containing N-terminal unique regions of these SFKs were run as a reference on the same gel. Positions and estimated amounts of SFKs are indicated on the right.
- Figure S2. Graphic presentation of densitometic data (JPG, 69.6 KB)
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Immunoblot and kinase data represented by Figures 4B, 4C, 4E, 4G and 5B were analyzed by densitometry. Error bars indicate SEMs. Asterisks indicate statistically significant differences between wt and hck−/−cells (P<.05 by Student t test).
- Figure S3. Effects of Hck deficiency on phosphorylation of ERK1/2 and Akt (JPG, 25.8 KB)
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IgE-sensitized wt and hck−/−cells were stimulated with 1 or 100 ng/mL DNP23-HSA for the indicated periods. Cell lysates were either directly analyzed by SDS-PAGE and immunoblotting with the indicated phospho-specific antibodies. The same blots were stripped of antibodies and reprobed with antibodies that detect the antigens irrespective of phosphorylation status.
- Figure S4. IgE + anti-IgE stimulation of wt and hck−/−mast cells (JPG, 52.1 KB)
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(A) IgE-sensitized wt and hck−/−cells were stimulated with 0, 2 or 20 µg/mL anti-IgE mAb B1E3 for 20 hours. TNF- and IL-6 secreted into culture supernatants were measured by ELISA. ND, not detected. Error bars represent SD. (B,C) IgE-sensitized wt and hck−/−cells were stimulated with 2 µg/mL anti-IgE mAb B1E3 for the indicated periods. Cell lysates were directly analyzed by SDS-PAGE followed by immunoblotting with antiphosphotyrosine mAb 4G10 or the indicated phospho-specific antibodies. The same blots were reprobed with antibodies that detect the antigens irrespective of phosphorylation status.
- Figure S5. Lyn and Hck show similar preferences towards different tyrosine residues in β-ITAM (JPG, 16.8 KB)
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In vitro kinase assays were performed using wt and mutant  -ITAM peptides, as described in Document S1. Error bars represent SD. Autophosphorylating activities of Lyn and Hck are shown below. Side bars indicate p56lyn, p56lyn, p59hck, and p56hck .
- Figure S6. Schematic of hierarchical regulation among SFKs and Fcε RImediated signaling under “high-intensity” and “low-intensity” stimulation conditions (JPG, 49.7 KB)
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(A) Hck inhibits Lyn activity and Lyn inhibits Fyn activity. Lyn plays positive and negative regulatory roles by phosphorylating the canonical and noncanonical tyrosine residues, respectively, in the -ITAM. Our present study indicates that Hck can phosphorylate the canonical tyrosine residues. It is not clear whether Hck can phosphorylate the noncanonical residue, Y-225. Although Fyn may be able to phosphorylate the canonical residues, there is no direct evidence yet. (B,C) Signaling events induced by Fc RI aggregation under “high-intensity” and “low-intensity” stimulation conditions. Major differences between the two conditions are that, under “high-intensity” stimulation conditions, Lyn plays a predominantly negative regulatory role by phosphorylating the noncanonical tyrosine residue in the -ITAM, which is linked to phosphorylation of negative signaling molecules such as SHIP and Dok1/2. However, increased activity of Lyn itself is not sufficient for phosphorylation of FcRI or SHIP, but “high-intensity” stimulation is essential for these events.
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