Dynamics of allosteric activation in PLC‐γ isozymes
Authors: Siraliev-Perez Edhriz , Hoffmann Reece , Stariha Jordan , Hajicek Nicole , Burke John , Sondek John 
Published: April 2020

The PLC‐γ isozymes (‐γ1, ‐γ2) hydrolyze the minor membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) into the second messengers diacylglycerol and inositol 1,4,5‐trisphosphate to control diverse biological processes including chemotaxis, vascular development, and immune function. In contrast, mutant forms of the PLC‐γ isozymes that are constitutively active promote autoimmune disorders and cancer. For example, PLC‐γ1 is the most frequently (~40%) mutated gene in patients with adult T‐cell leukemia/lymphoma. However, despite widespread appreciation of the PLC‐γ isozymes in diverse biological processes, their regulation remains poorly understood.We recently determined the crystal structure of full‐length PLC‐γ1 at high resolution. The structure highlights a conserved catalytic core blocked from accessing membranes by a regulatory array unique to the PLC‐γ isozymes. Within the regulatory array, an N‐terminal SH2 (nSH2) domain is arranged to engage phosphorylated portions of tyrosine kinases. Complex formation facilitates the recruitment of PLC‐γ1 to membranes and the phosphorylation of Tyr783, also within the regulatory array. In contrast to the nSH2 domain, the equivalent surface of the C‐terminal SH2 (cSH2) domain is blocked by interactions with the catalytic core. Nonetheless, this surface must bind phosphorylated Tyr783 for lipase activation.We hypothesize that engagement of a kinase to the nSH2 domain propagates structural alterations into the cSH2 domain that destabilize its interactions with the catalytic core thus priming it to bind phosphorylated Tyr783. This interaction presumably leads to a larger rearrangement of the regulatory array relative to the catalytic core necessary for membrane engagement and PIP2 hydrolysis. This hypothesis is supported by alterations highlighted in the structural comparison of full‐length PLC‐γ1 with a pre‐existing structure of the two SH2 domains bound to the kinase domain of fibroblast growth factor receptor 1 (FGFR1K). To test this hypothesis, we used a fluorogenic PIP2 analog and hydrogen‐deuterium exchange coupled to mass spectrometry to assess activity and structural changes in PLC‐γ1 upon engagement of FGFR1K, respectively.Here we show that binding of FGFR1K to PLC‐γ1 increases deuterium incorporation within the interface between the regulatory array and the catalytic core suggesting that interaction of FGFR1K and PLC‐γ1 disrupts autoinhibition. The addition of liposomes further increases deuterium incorporation within the same region suggesting that membranes reinforce this disruption. Similar, but more dramatic increases in deuterium incorporation were observed for the cancer‐associated and constitutively active variant, PLC‐γ1 (D1165H). Finally, we show that a two‐fold molar excess of FGFR1K relative to PLC‐γ1 increased lipase activity up to three‐fold. Taken together, these data strongly suggest that kinases shift the conformational equilibrium of PLC‐γ isozymes to facilitate access and hydrolysis of membrane‐resident PIP2. This allosteric effect is recapitulated by oncogenic mutations and reinforced by membranes.Support or Funding InformationThis work was supported by NIH Grant R01‐GM057391 (J.S.). E.S‐P. was supported by an NSF GRF under Grant DGE‐1650116.