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. Author manuscript; available in PMC: 2018 May 17.
Published in final edited form as: Biochemistry. 2017 May 25;56(23):2938–2949. doi: 10.1021/acs.biochem.6b01182

Figure 6. PIP3 activates ITK and the activation loop in the ITK kinase domain is sterically blocked by the PHTH domain in autoinhibted ITK.

Figure 6

(a) In vitro ITK kinase assay (500 nM ITK) in solution (lane 1), in the presence of PIP3 containing liposomes (lane 2) and control liposomes that lack PIP3 (lane 3). PC indicates 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and PS is 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS). ITK activation loop autophosphorylation (pY511) is detected at 30 minutes using anti-BTK pY551 and total enzyme levels are detected using anti-ITK antibody. (b) Histogram representation of normalized pY511 levels from the ITK kinase assays shown in (a). Normalization was done as in Figure 4(c and d), except that the value for ITK in the absence of any phosphoinositol compound was set to 1. Experiments were run in triplicate. (c) Domain constructs of ITK and LCK. Full length ITK and the fragment lacking the PHTH domain (ITK SH3-SH2-Kinase) are FLAG-tagged at the C-termini and the LCK kinase domain carries a His6 tag at the N-terminus. Full length ITK and the ITK SH3-SH2-kinase fragment contain the K390R mutation rendering both inactive24. (d) In vitro LCK kinase assay in solution (lanes 3 and 4), in the presence of PIP3 containing liposomes (lanes 5 and 6) and control liposomes that lack PIP3 (lanes 7 and 8). ITK SH3-SH2-Kinase (K390R) (1 μM, lanes 3,5,7) and full length ITK (K390R) (1 μM, lanes 4,6,8) serve as substrates for LCK (200 nM). Lanes 1 and 2 are no enzyme (LCK) controls. Phosphorylation of ITK Y511 is detected using anti-BTK pY551 and total substrate and enzyme levels are detected using anti-FLAG and anti-His6 antibodies, respectively. (e) Histogram representation of normalized pY511 levels from the LCK kinase assays shown in (d). Normalization as in panel (b) with the value for full length ITK (K390R) in the absence of liposomes set to 1. Data for the ITK SH3-SH2-Kinase (K390R) substrate is on the left and the full length ITK (K390R) substrate is on the right. Experiments were run in triplicate.