The activation loop in Lck regulates oncogenic potential by inhibiting basal kinase activity and restricting substrate specificity

Laham, Lorraine E.; Mukhopadhyay, Nishit K.; Roberts, Thomas M.

doi:10.1038/sj.onc.1203738

Cited by 16 publications

(14 citation statements)

References 36 publications

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“…In a structure of the tyrosine kinase, Lck, where the kinase domain adopts an active conformation, a leucine residue occupies this position in the activation loop and interacts with a similar hydrophobic pocket ( Fig 6C ) (PDB 3LCK). Mutation of this leucine residue in Lck to an aspartic acid residue results in a less active Lck variant, which is consistent with observations for FLT3 [ 37 ]. Stabilizing the active conformation of FLT3 in this fashion would disfavor quizartinib binding since the drug recognizes an inactive conformation.…”

Section: Resultssupporting

confidence: 90%

Crystal Structure of the FLT3 Kinase Domain Bound to the Inhibitor Quizartinib (AC220)

et al. 2015

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More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

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Section: Resultssupporting

confidence: 90%

Crystal Structure of the FLT3 Kinase Domain Bound to the Inhibitor Quizartinib (AC220)

et al. 2015

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“…6, Fig. S5), which has been shown to render Lck kinase-dead 37 . Different diffusion coefficients of 0.82 μm 2 s -1 (0.81-0.83) and 1.13 μm 2 s -1 (1.12-1.15) were observed for Lck K273R -PAmCherry in stimulated and resting cells, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

Conformational states control Lck switching between free and confined diffusion modes in T cells

Hilzenrat

Pandžić²,

Yang

et al. 2018

Preprint

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17T cell receptor (TCR) phosphorylation by Lck is an essential step in T cell activation. It is 18 known the conformational states of Lck control enzymatic activity; however, the underlying 19 principles of how Lck finds its substrate in the plasma membrane remain elusive. Here, 20 single-particle tracking is paired with photoactivatable localization microscopy (sptPALM) to 21 observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules 22 switched between free and confined diffusion in resting and stimulated T cells. 23 Conformational state, but not partitioning into membrane domains, caused Lck confinement 24 as open conformation Lck was more confined than closed. Further confinement of kinase-25 dead versions of Lck suggests that Lck interacts with open active Lck to cause confinement, 26 irrespectively of kinase activity. Our data supports a model that confined diffusion of open 27Lck results in high local phosphorylation rates and closed Lck diffuses freely to enable wide-28 range scanning of the plasma membrane. 29T cell signaling is a tightly controlled process involving both simultaneous and sequential 30 spatiotemporal events, involving membrane remodeling and redistribution of key signaling 31 proteins 1,2 . Engagement of the T cell receptor (TCR) with an antigenic pMHC on the surface 32 of an antigen-presenting cell (APC) leads to the formation of immunological synapses 3 and 33 initiates downstream signaling events that lead to T cell activation 4 . The Src family kinase 34 Lck plays a crucial role in the signaling cascade. TCR engagement results in the membrane 35 release 5 and phosphorylation of the immunoreceptor tyrosine-based motifs (ITAMs) located 36 in the cytoplasmic tails of the CD3ζ chain by Lck 6 . Phosphorylated sites on the TCR-CD3 37 complex become docking sites for the zeta chain-associated protein kinase 70 (ZAP70), that 38 is further phosphorylated by Lck 7 before recruiting other proteins in the signaling cascade 39 that are necessary for complete T cell activation. 40The role of Lck in T cell activation as a signaling regulator is of particular interest due to its 41 dynamic characteristics. Lck is a 56 kDa protein comprised of a Src homology (SH) 4 domain 42 at the N-terminus, followed by a unique domain, an SH3 domain, an SH2 domain, a kinase 43 domain and short C-terminal tail. Lck is anchored to the plasma membrane through its SH4 44 domain via post-translational acylation on three specific sites: a myristoylated Gly2 8 and 45 palmitoylated Cys3 and Cys5. The latter two are crucial for membrane binding and biological 46 activity, enabling Lck diffusion in the inner leaflet of the plasma membrane and its 47 recruitment to the immunological synapse 9 . The unique domain interacts with the CD3ε 48 subunit in the TCR-CD3 complex 10 as well as the co-receptors CD4 and CD8 11 via zinc-49 mediated bonds. However, Lck does not require the co-receptors for recruitment to the 50 immunological synapse or for TCR triggering 12 , suggesting that freely diffusing Lck is...

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“…1). The enhanced phosphorylation of p56/Lck was due to phosphorylation of Y394, which is analogous to Src Y416 and associated with the open, active conformation of p56/Lck (31). In contrast, phosphorylation of Y505, which is analogous to Src Y527 and is phosphorylated in the closed inactive form of p56/Lck, decreased by 85% (Fig.…”

Section: Resultsmentioning

confidence: 99%

Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis

Betapudi

Shukla²,

Alluri³

et al. 2016

FASEB j.

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Previous studies have demonstrated that cleaved high-molecular-weight kininogen (HKa) induces endothelial apoptosis and inhibits angiogenesis and have suggested that this occurs through inhibition of Src family kinases. This study assessed the role of tyrosine-protein kinase Lck (p56/Lck) in this pathway. We analyzed early events leading to apoptosis of human endothelial cells exposed to HKa. The role of p56/Lck was investigated using short interfering (si) RNA knockdown and lentivirus expression in assays of endothelial tube formation, sprouting of neovessels from murine aorta, and angiogenesis in Matrigel plugs. HKa stimulated expression and phosphorylation of p56/Lck. siRNA knockdown of p56/Lck promoted endothelial proliferation and blocked HKa-induced apoptosis and activation of p53, Bax, and Bak. Lentivirus expression of p56/Lck in endothelial cells induced apoptosis and blocked tube formation. Expression of p56/Lck in murine aortic rings blocked sprouting angiogenesis. Lentivirus expressing p56/Lck blocked angiogenesis in Matrigel plugs, while p56/Lck short hairpin RNA inhibited the antiangiogenic effect of HKa. Scrambled siRNAs and empty lentiviral vectors were used in all experiments. Apoptosis of proliferating endothelial cells and inhibition of angiogenesis by HKa requires p56/Lck. This suggests a novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.-Betapudi, V., Shukla, M., Alluri, R., Merkulov, S., McCrae, K. R. Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.

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The activation loop in Lck regulates oncogenic potential by inhibiting basal kinase activity and restricting substrate specificity

Cited by 16 publications

References 36 publications

Crystal Structure of the FLT3 Kinase Domain Bound to the Inhibitor Quizartinib (AC220)

Crystal Structure of the FLT3 Kinase Domain Bound to the Inhibitor Quizartinib (AC220)

Conformational states control Lck switching between free and confined diffusion modes in T cells

Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis

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