Phase separation of a PKA regulatory subunit controls cAMP compartmentation and oncogenic signaling

. Author manuscript; available in PMC: 2021 Sep 17.

Published in final edited form as: Cell. 2020 Aug 25;182(6):1531–1544.e15. doi: 10.1016/j.cell.2020.07.043

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and Virus Strains
DH5a competent bacteria	NEB	Cat# C2987I
RIα-EGFP lentivirus	This paper	N/A
RIα_D/D+Lmker-EGFP lentivirus	This paper	N/A
RIα_{Δ(D/D+Linker)}-EGFP lentivirus	This paper	N/A
BL21(DE3) competent bacteria	Agilent	Cat# 200131
Rosetta (DE3) pLysS competent bacteria	Millipore Sigma	Cat# 70956
Biological Samples
Sprague-Dawley rat pups	Charles River	Strain Code 400
Chemicals, Peptides, and Recombinant Proteins
BbsI	NEB	Cat# R3539
Q5 High Fidelity Polymerase	NEB	Cat# M0491S
HiFi DNA Assembly Kit	NEB	Cat# E5520S
HindIII	NEB	Cat# R0104S
EcoRI	NEB	Cat# R0101S
XbaI	NEB	Cat# R0145S
Q5 Site Directed Mutagenesis Kit	NEB	Cat# E0554
DMEM:F12	ThermoFisher	Cat# 12634010
ITS Liquid Media Supplement	Sigma Aldrich	Cat# I3146
Dexamethasone	Sigma Aldrich	Cat# D1159
Polyjet	Signagen	Cat# SL100688
DNase I	ThermoFisher	Cat# EN0525
Neurobasal media	ThermoFisher	Cat# 21103049
SM1 Supplement	STEMCELL Technologies	Cat# 05711
Poly-D-Lysine	Sigma Aldrich	Cat# P6407
Lipofectamine LTX	Invitrogen	Cat# 15338500
Puromycin	Sigma Aldrich	Cat# P8833
BSA	Roche	Cat# 10738328103
HEPES	Sigma Aldrich	Cat# H3375
EDTA	Sigma Aldrich	Cat# E6758
DAPI	ThermoFisher	Cat# D21490
Noble Agar	ThermoFisher	Cat# AAJ1090722
Powdered DMEM	ThermoFisher	Cat# 12800017
IPTG	Sigma Aldrich	Cat# I6758
MES	Sigma Aldrich	Cat# M3671
EGTA	Sigma Aldrich	Cat# E3889
DTT	Sigma Aldrich	Cat# D0632
Protease inhibitors	Roche	Cat# 11873580001
cGMP	Sigma Aldrich	Cat# G7504
β-mercaptoethanol	Sigma Aldrich	Cat# M6250
Imidazole	Sigma Aldrich	Cat# 1336500
ATP	Sigma Aldrich	Cat# A26209
cAMP	Sigma Aldrich	Cat# A9501
PEG4000	Sigma Aldrich	Cat# 1546569
Forskolin	CalBioChem	Cat# 344281
IBMX	Sigma Aldrich	Cat# I7018
Rolipram	Alexis	Cat# 61413-54-5
1,6-hexanediol	Sigma Aldrich	Cat# 240117
Myr-PKI	Tocris	Cat# 2546
Isoproterenol	Sigma Aldrich	Cat# 1351005
Critical Commercial Assays
BrdU kit	Invitrogen	Cat# B23151
MDL-12330A	Sigma Aldrich	Cat# M182
8-Br-2’-O-Me-cAMP-AM	Biolog	Cat# B028–01
8-[Φ−450]-cAMP	Biolog	Cat# P024–001
Deposited Data
FluoSTEP-ICUE sequence	GenBank	MT800777.1
FluoSTEP-AKAR sequence	GenBank	MT800778.1
Experimental Models: Cell Lines
HEK293T	ATCC	ATTC Cat# CRL-11268
HEK293A	ThermoFisher	Cat# R70507
MEF	GS McKnight Lab, University of Washington, Seattle, WA, USA	N/A
AML12	ATCC	ATCC Cat# CRL-2254
293-RIα	This paper	N/A
293T-RIα KO	This paper	N/A
MEF-RIα KO	(Day et al., 2011)	N/A
AML12-RIα KO	This paper	N/A
AML 12-RIα KO-RIα-EGFP	This paper	N/A
AML12-RIα KO_D/D+Linker-RIα-EGFP	This paper	N/A
AML12-RIα KO_{Δ(D/D+Linker)}-RIα-EGFP	This paper	N/A
MEF-RIα KO-RIα-EGFP	This paper	N/A
MEF-RIα KO-RIα_D/D+Linker-EGFP	This paper	N/A
MEF-RIα KO-RIα_{Δ(D/D+Linker)}-EGFP	This paper	N/A
Oligonucleotides
Primers for plasmid construction, see Table S1	This paper	N/A
RIα-FP₁₁ HDR ssDNA, see Table S1	IDT, for this paper	N/A
DnaJB1 exon 1 gBlock, see Table S1	IDT, for this paper	N/A
Recombinant DNA
px458	(Ran et al., 2013)	Addgene plasmid #48138
px459	(Ran et al., 2013)	Addgene plasmid #62988
px458 mouse RIα gRNA	This paper	N/A
px459 human RIα gRNA	This paper	N/A
pcDNA3 AKAR2-CR	(Lam et al., 2012)	Addgene plasmid #40255
pBAD-mT agBFP2	(Subach et al., 2011)	Addgene plasmid #34632
pcDNA3.1 EGFP-RIα	(Day et al., 2011)	N/A
pcDNA3.1 mCherry-PKA_cat	(Day et al., 2011)	N/A
pcDNA3.1 mRuby2-RIα	This paper	N/A
pcDNA3.1 mTagBFP2-RIα	This paper	N/A
pcDNA3.1 RIα_D/D+Linker-EGFP	This paper	N/A
pcDNA3.1 RIα_D/D-EGFP	This paper	N/A
pcDNA3.1 RIα_Linker-EGFP	This paper	N/A
pcDNA3.1 RIα_{Δ(D/D+Linker)}-EGFP	This paper	N/A
pcDNA3.1 RIα_ΔD/D-EGFP	This paper	N/A
pcDNA3.1 RIα_ΔLinker-EGFP	This paper	N/A
pcDNA3.1 mTagBFP2-PKA_cat	This paper	N/A
pcDNA3.1 GFP_1–10	(Leonetti et al., 2016)	Addgene plasmid #70219
pcDNA3 AKAR4	(Depry et al., 2011)	Addgene plasmid #61619
pcDNA3 ICUE3	(DiPilato and Zhang, 2009)	Addgene plasmid #61622
pcDNA3.1 FluoSTEP-AKAR	This paper	N/A
pcDNA3.1 FluoSTEP-ICUE	This paper	N/A
pcDNA3.1 Fluo STEP-AKAR(T/A)	This paper	N/A
pcDNA3.1 Fluo STEP-ICUE(R279E)	This paper	N/A
pcDNA3.1 ICUE4	This paper	N/A
PDE4D2 cDNA	gift from Hengming Ke, UNC, Chapel Hill, NC	N/A
pcDNA3.1 PDE4D2_cat-ICUE4	This paper	N/A
pcDNA3.1 RIα-ICUE4	This paper	N/A
pcDNA3.1 RIα_D/D+Linker-ICUE4	This paper	N/A
pcDNA3.1 RG-ICUE	This paper	N/A
pcDNA3.1 RIα-RG-ICUE	This paper	N/A
pcDNA3.1 mCherry-DnaJB 1-PKAc_at	This paper	N/A
pcDNA3.1 mCherry-DnaJB1-PKA_cat^K72H	This paper	N/A
pcDNA3.1 mTagBFP2-DnaJB1-PKA_cat	This paper	N/A
pcDNA3.1 mT agBFP2-DnaJB 1-PKA_cat^K72H	This paper	N/A
pcDNA3.1 mCherry-PKA_cat^G1A	This paper	N/A
pcDNA3.1 mCherry-Myr-DnaJB1-PKA_cat	This paper	N/A
pcDNA3.1 mCherry-DnaJB 1-PKA_cat	This paper	N/A
pcDNA3.1 mCherry-DnaJB1^H33Q-PKA_cat	This paper	N/A
pcDNA3.1 mCherry-Myr-DnaJB 1 ^H33Q-PKA_cat	This paper	N/A
pLenti-puro	(Guan et al., 2011)	Addgene plasmid #39481
pLenti RIα-EGFP	This paper	N/A
pLenti RIα_D/D+Linker-EGFP	This paper	N/A
pLenti RIα_{Δ(D/D+Linker)}-EGFP	This paper	N/A
pRSET B sfGFP	This paper	N/A
pRSET B	Invitrogen	Cat# V35120
pEvolvR-enCas9-PolI3M-TBD	(Halperin et al., 2018)	Addgene plasmid #113077
pMD2.G	(Dull et al., 1998)	Addgene plasmid #12259
psPAX2	(Dull et al., 1998)	Addgene plasmid #12260
pET-His6-SUMO-TEV-LIC-PKA_cat	(Lu et al., 2019)	N/A
pET-His6-SUMO-TEV-LIC-DnaJB 1-PKA_cat	(Lu et al., 2019)	N/A
pET-His6-EGFP-PKA_cat	(Lu et al., 2019)	N/A
Software and Algorithms
MATLAB	MathWorks	https://www.mathworks.com/products/matlab.html
PRISM	Graphpad	https://www.graphpad.com/scientific-software/prism/
Adobe Illustrator	Adobe	https://www.adobe.com/products/illustrator
ImageJ	NIH	https://imagej.nih.gov