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Light-gated integrator for highlighting kinase activity in living cells

ABSTRACT: Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into “permanent” fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE272741 | GEO | 2024/07/27

REPOSITORIES: GEO

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Project description:The pattern of gene transcription in Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. To investigate the requirement of PKA for glucose control of gene expression, we have analyzed global transcription in strains devoid of PKA activity. In S. cerevisiae three genes, TPK1, TPK2, TPK3, encode catalytic subunits of PKA and the triple mutant tpk1 tpk2 tpk3 is unviable. We have worked, therefore, with two strains, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4, that bear suppressor mutations,. We have identified different classes of genes that can be induced, or repressed, by glucose in the absence of PKA. Among these genes, some are also controlled by a redundant signalling pathway involving PKA activation, while others do not respond to an increase in cAMP concentration. On the other hand, among genes which do not respond to glucose in the absence of PKA, some show a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. The goal of the present study was to investigate the occurrence of PKA-independent glucose signalling in S. cerevisiae. To this end, we have used global transcription analysis to study the effects of glucose on yeast strains completely devoid of PKA activity. In S. cerevisiae three genes TPK1, TPK2,and TPK3 encode catalytic subunits of PKA. While strains expressing only one of these genes grow normally, a triple null mutant (tpk1 tpk2 tpk3) is not viable (Toda et al 1987). Identification of different mutations able to suppress the growth defect of the triple mutant (Garrett and Broach 1989, Reinders et al 1998, Smith et al 1998) has allowed to determine what is the crucial function of PKA. As shown in Fig.1, PKA is needed to counteract the negative effect of the protein kinase Yak1 on yeast growth (Hartley et al 1994, Moriya et al 2001). In the presence of PKA the protein kinase Rim15 (Reinders et al 1998) and the transcription factors Msn2 and Msn4 (Görner et al 1998) can be phosphorylated and exported to the cytoplasm, transcription of the YAK1 gene, which is activated by Msn2/Msn4 (Smith et al 1998), is reduced, Yak1 levels remain low and growth is not hindered. In the absence of PKA, Rim15 remains in the nucleus where it can activate Msn2/Msn4 (Cameroni et al 2004) that turn on YAK1 transcription, thus blocking growth. This explains why strains lacking Rim15, Msn2/Msn4 or Yak1 no longer require PKA for growth. In this work we have used two isogenic strains lacking PKA and carrying the suppressor mutations msn2 msn4 or yak1. Two different suppressor mutants were used with the aim to enable a dissection of effects of the lack of PKA and effects of the suppressor mutations themselves.

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Light-gated integrator for highlighting kinase activity in living cells

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