Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.

The DNAJ-PKAc fusion kinase is a defining feature of fibrolamellar carcinoma (FLC). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. By combining proximity proteomics, biochemical analyses, and live-cell photoactivation microscopy, we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates, including proteins involved in translation and the anti-apoptotic factor Bcl-2-associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Tissue samples from patients with FLC exhibit increased levels of BAG2 in primary and metastatic tumors. Furthermore, drug studies implicate the DNAJ-PKAc/Hsp70/BAG2 axis in potentiating chemotherapeutic resistance. We find that the Bcl-2 inhibitor navitoclax enhances sensitivity to etoposide-induced apoptosis in cells expressing DNAJ-PKAc. Thus, our work indicates BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.

HAF Prevents Hepatocyte Apoptosis and Hepatocellular Carcinoma through Transcriptional Regulation of the NF-κB pathway.

Background: Hepatocellular carcinoma (HCC) incidence is increasing worldwide due to the obesity epidemic, which drives metabolic dysfunction-associated steatohepatitis (MASH) that can lead to HCC. However, the molecular pathways that lead to MASH-HCC are poorly understood. We have previously reported that male mice with global haploinsufficiency of hypoxia-associated factor, HAF ( SART1 +/ - ) spontaneously develop MASH/HCC. However, the cell type(s) responsible for HCC associated with HAF loss are unclear. Results: SART1 -floxed mice were crossed with mice expressing Cre-recombinase within hepatocytes (Alb-Cre; hepS -/- ) or macrophages (LysM-Cre, macS -/- ). Only hepS -/- mice (both male and female) developed HCC suggesting that HAF protects against HCC primarily within hepatocytes. HAF-deficient macrophages showed decreased P-p65 and P-p50 and in many major components of the NF-κB pathway, which was recapitulated using HAF siRNA in vitro . HAF depletion increased apoptosis both in vitro and in vivo , suggesting that HAF mediates a tumor suppressor role by suppressing hepatocyte apoptosis. We show that HAF regulates NF-κB activity by controlling transcription of TRADD and RIPK1 . Mice fed a high-fat diet (HFD) showed marked suppression of HAF, P-p65 and TRADD within their livers after 26 weeks, but manifest profound upregulation of HAF, P-65 and TRADD within their livers after 40 weeks of HFD, implicating deregulation of the HAF-NF-κB axis in the progression to MASH. In humans, HAF was significantly decreased in livers with simple steatosis but significantly increased in HCC compared to normal liver. Conclusions: HAF is novel transcriptional regulator of the NF-κB pathway that protects against hepatocyte apoptosis and is a key determinant of cell fate during progression to MASH and MASH-HCC.

Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.

The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Immunoblot and immunohistochemical analyses of FLC patient samples correlate increased levels of BAG2 with advanced disease and metastatic recurrences. BAG2 is linked to Bcl-2, an anti-apoptotic factor that delays cell death. Pharmacological approaches tested if the DNAJ- PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in AML12 DNAJ-PKAc hepatocyte cell lines using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12 DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a biomarker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.

Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.

Dynamic changes in protein-protein interaction (PPI) networks underlie all physiological cellular functions and drive devastating human diseases. Profiling PPI networks can, therefore, provide critical insight into disease mechanisms and identify new drug targets. Kinases are regulatory nodes in many PPI networks; yet, facile methods to systematically study kinase interactome dynamics are lacking. We describe kinobead competition and correlation analysis (kiCCA), a quantitative mass spectrometry-based chemoproteomic method for rapid and highly multiplexed profiling of endogenous kinase interactomes. Using kiCCA, we identified 1,154 PPIs of 238 kinases across 18 diverse cancer lines, quantifying context-dependent kinase interactome changes linked to cancer type, plasticity, and signaling states, thereby assembling an extensive knowledgebase for cell signaling research. We discovered drug target candidates, including an endocytic adapter-associated kinase (AAK1) complex that promotes cancer cell epithelial-mesenchymal plasticity and drug resistance. Our data demonstrate the importance of kinase interactome dynamics for cellular signaling in health and disease.

Proximity biotinylation to define the local environment of the protein kinase A catalytic subunit in adrenal cells.

Mutant protein kinase A catalytic subunit (PKAc) drives adrenal Cushing's syndrome, though its signaling interactions remain unclear. This protocol details steps to use live-cell proximity labeling to identify subcellular compartments and proteins closely associated with variants of PKAc in human adrenal cells. We include instructions for clonal cell line generation, live biotin labeling of proximal proteins, isolation of biotinylated proteins, and sample processing for proteomic analysis using the biotin ligase miniTurbo with wild-type and mutant PKAc.1,2 For complete details on the use and execution of this protocol, please refer to Omar et al. (2022).3.

Mislocalization of protein kinase A drives pathology in Cushing's syndrome.

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals.

KiRNet: Kinase-centered network propagation of pharmacological screen results.

The ever-increasing size and scale of biological information have popularized network-based approaches as a means to interpret these data. We develop a network propagation method that integrates kinase-inhibitor-focused functional screens with known protein-protein interactions (PPIs). This method, dubbed KiRNet, uses an a priori edge-weighting strategy based on node degree to establish a pipeline from a kinase inhibitor screen to the generation of a predictive PPI subnetwork. We apply KiRNet to uncover molecular regulators of mesenchymal cancer cells driven by overexpression of Frizzled 2 (FZD2). KiRNet produces a network model consisting of 166 high-value proteins. These proteins exhibit FZD2-dependent differential phosphorylation, and genetic knockdown studies validate their role in maintaining a mesenchymal cell state. Finally, analysis of clinical data shows that mesenchymal tumors exhibit significantly higher average expression of the 166 corresponding genes than epithelial tumors for nine different cancer types.

Phosphoproteomic Analysis as an Approach for Understanding Molecular Mechanisms of cAMP-Dependent Actions.

In recent years, highly sensitive mass spectrometry-based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cAMP. Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This minireview describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors' laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone-producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate-limiting step likely regulate these and, by analogy, many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists. SIGNIFICANCE STATEMENT: Phosphoproteomic analyses identify thousands of altered phosphorylation sites upon drug treatment, providing many possible regulatory targets but also highlighting questions about which phosphosites are functionally important. These data imply that multistep processes are regulated by phosphorylation at not one but rather many sites. Most previous studies assumed a single step or very few rate-limiting steps were changed by phosphorylation. This concept should be changed. Previous interpretations also assumed substoichiometric phosphorylation was not of regulatory importance. This assumption also should be changed.

Pharmacoproteomics Identifies Kinase Pathways that Drive the Epithelial-Mesenchymal Transition and Drug Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a complex and deadly disease lacking druggable genetic mutations. The limited efficacy of systemic treatments for advanced HCC implies that predictive biomarkers and drug targets are urgently needed. Most HCC drugs target protein kinases, indicating that kinase-dependent signaling networks drive HCC progression. To identify HCC signaling networks that determine responses to kinase inhibitors (KIs), we apply a pharmacoproteomics approach integrating kinome activity in 17 HCC cell lines with their responses to 299 KIs, resulting in a comprehensive dataset of pathway-based drug response signatures. By profiling patient HCC samples, we identify signatures of clinical HCC drug responses in individual tumors. Our analyses reveal kinase networks promoting the epithelial-mesenchymal transition (EMT) and drug resistance, including a FZD2-AXL-NUAK1/2 signaling module, whose inhibition reverses the EMT and sensitizes HCC cells to drugs. Our approach identifies cancer drug targets and molecular signatures of drug response for personalized oncology.

Kinobead/LC-MS Phosphokinome Profiling Enables Rapid Analyses of Kinase-Dependent Cell Signaling Networks.

Kinase-catalyzed protein phosphorylation is fundamental to eukaryotic signal transduction, regulating most cellular processes. Kinases are frequently dysregulated in cancer, inflammation, and degenerative diseases, and because they can be inhibited with small molecules, they became important drug targets. Accordingly, analytical approaches that determine kinase activation states are critically important to understand kinase-dependent signal transduction and to identify novel drug targets and predictive biomarkers. Multiplexed inhibitor beads (MIBs or kinobeads) efficiently enrich kinases from cell lysates for liquid chromatography-mass spectrometry (LC-MS) analysis. When combined with phosphopeptide enrichment, kinobead/LC-MS can also quantify the phosphorylation state of kinases, which determines their activation state. However, an efficient kinobead/LC-MS kinase phospho-profiling protocol that allows routine analyses of cell lines and tissues has not yet been developed. Here, we present a facile workflow that quantifies the global phosphorylation state of kinases with unprecedented sensitivity. We also found that our kinobead/LC-MS protocol can measure changes in kinase complex composition and show how these changes can indicate kinase activity. We demonstrate the utility of our approach in specifying kinase signaling pathways that control the acute steroidogenic response in Leydig cells; this analysis establishes the first comprehensive framework for the post-translational control of steroid biosynthesis.

Targeting Dynamic ATP-Binding Site Features Allows Discrimination between Highly Homologous Protein Kinases.

ATP-competitive inhibitors that demonstrate exquisite selectivity for specific members of the human kinome have been developed. Despite this success, the identification of highly selective inhibitors is still very challenging, and it is often not possible to rationally engineer selectivity between the ATP-binding sites of kinases, especially among closely related family members. Src-family kinases (SFKs) are a highly homologous family of eight multidomain, nonreceptor tyrosine kinases that play general and specialized roles in numerous cellular processes. The high sequence and functional similarities between SFK members make it hard to rationalize how selectivity can be gained with inhibitors that target the ATP-binding site. Here, we describe the development of a series of inhibitors that are highly selective for the ATP-binding sites of the SFKs Lyn and Hck over other SFKs. By biochemically characterizing how these selective ATP-competitive inhibitors allosterically influence the global conformation of SFKs, we demonstrate that they most likely interact with a binding pocket created by the movement of the conformationally flexible helix αC in the ATP-binding site. With a series of sequence swap experiments, we show that sensitivity to this class of selective inhibitors is due to the identity of residues that control the conformational flexibility of helix αC rather than any specific ATP-binding site interactions. Thus, the ATP-binding sites of highly homologous kinases can be discriminated by targeting heterogeneity within conformationally flexible regions.

An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric enzyme consisting of a chaperonin-binding domain fused to the Cα subunit of protein kinase A. Biochemical analyses of clinical samples reveal that a unique property of this fusion enzyme is the ability to recruit heat shock protein 70 (Hsp70). This cellular chaperonin is frequently up-regulated in cancers. Gene-editing of mouse hepatocytes generated disease-relevant AML12DNAJ-PKAc cell lines. Further analyses indicate that the proto-oncogene A-kinase anchoring protein-Lbc is up-regulated in FLC and functions to cluster DNAJ-PKAc/Hsp70 sub-complexes with a RAF-MEK-ERK kinase module. Drug screening reveals Hsp70 and MEK inhibitor combinations that selectively block proliferation of AML12DNAJ-PKAc cells. Phosphoproteomic profiling demonstrates that DNAJ-PKAc biases the signaling landscape toward ERK activation and engages downstream kinase cascades. Thus, the oncogenic action of DNAJ-PKAc involves an acquired scaffolding function that permits recruitment of Hsp70 and mobilization of local ERK signaling.

A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Depletion of dAKAP1-protein kinase A signaling islands from the outer mitochondrial membrane alters breast cancer cell metabolism and motility.

Breast cancer screening and new precision therapies have led to improved patient outcomes. Yet, a positive prognosis is less certain when primary tumors metastasize. Metastasis requires a coordinated program of cellular changes that promote increased survival, migration, and energy consumption. These pathways converge on mitochondrial function, where distinct signaling networks of kinases, phosphatases, and metabolic enzymes regulate these processes. The protein kinase A-anchoring protein dAKAP1 compartmentalizes protein kinase A (PKA) and other signaling enzymes at the outer mitochondrial membrane and thereby controls mitochondrial function and dynamics. Modulation of these processes occurs in part through regulation of dynamin-related protein 1 (Drp1). Here, we report an inverse relationship between the expression of dAKAP1 and mesenchymal markers in breast cancer. Molecular, cellular, and in silico analyses of breast cancer cell lines confirmed that dAKAP1 depletion is associated with impaired mitochondrial function and dynamics, as well as with increased glycolytic potential and invasiveness. Furthermore, disruption of dAKAP1-PKA complexes affected cell motility and mitochondrial movement toward the leading edge in invasive breast cancer cells. We therefore propose that depletion of dAKAP1-PKA "signaling islands" from the outer mitochondrial membrane augments progression toward metastatic breast cancer.

Kinome chemoproteomics characterization of pyrrolo[3,4-c]pyrazoles as potent and selective inhibitors of glycogen synthase kinase 3.

Glycogen synthase kinase 3 has evolutionarily conserved roles in cell signaling and metabolism and is a recognized drug target in neurological pathologies, most prominently bipolar disorder. More recently it has been suggested that GSK3 may be a target for the treatment of trypanosomatid parasite infections, e.g. with T. brucei, due to the lethal phenotype observed in parasite GSK3 short RNAi knockdown experiments. Here we investigated the kinome selectivity of a library of pyrrolo[3,4-c]pyrazol inhibitors that were developed against T. brucei GSK3 but that also interact with the human orthologue and other protein kinases. We applied label-free MS-based kinome chemoproteomics profiling with kinobeads to obtain the selectivity profiles of all 39 library members against 217 human protein and lipid kinases. This allowed us to study the structure-activity relationship of the library members as well as the chemical genetic relationships between kinase targets. As a result, we identified a novel and highly selective HsGSK3 inhibitor containing a 2-chloroaniline-substituted squaric acid amide pharmacophore that confers low nanomolar (IC50 = 2.8 nM) and sub-micromolar potency against purified and cellular HsGSK3. The inhibitor will be useful as a new lead for GSK3 inhibitor development and as a chemical genetic probe to study roles of GSK3 in cell signaling.

7 H-Pyrrolo[2,3- d]pyrimidin-4-amine-Based Inhibitors of Calcium-Dependent Protein Kinase 1 Have Distinct Inhibitory and Oral Pharmacokinetic Characteristics Compared with 1 H-Pyrazolo[3,4- d]pyrimidin-4-amine-Based Inhibitors.

Selective inhibitors of Cryptosporidium calcium-dependent protein kinase 1 ( CpCDPK1) based on the 1 H-pyrazolo[3,4- d]pyrimidin-4-amine (pyrazolopyrimidine, PP) scaffold are effective in both in vitro and in vivo models of cryptosporidiosis. However, the search for distinct safety and pharmacokinetic (PK) properties has motivated our exploration of alternative scaffolds. Here, we describe a series of 7 H-pyrrolo[2,3- d]pyrimidin-4-amine (pyrrolopyrimidine, PrP)-based analogs of PP CpCDPK1 inhibitors. Most of the PrP-based inhibitors described potently inhibit the CpCDPK1 enzyme, demonstrate no toxicity against mammalian cells, and block proliferation of the C. parvum parasite in the low micromolar range. Interestingly, certain substituents that show reduced CpCDPK1 potency when displayed from a PP scaffold provided notably enhanced efficacy in the context of a PrP scaffold. PK studies on these paired compounds show that some PrP analogs have distinct physiochemical properties compared with their PP counterparts. These results demonstrate that inhibitors based on a PrP scaffold are distinct therapeutic alternatives to previously developed PP inhibitors.

Proteomic Profiling of Protein Kinase Inhibitor Targets by Mass Spectrometry.

Identifying cellular targets of bioactive small molecules from large-scale screening campaigns can be a significant bottleneck in developing novel therapeutics. Our rapid small-molecule target profiling protocol combines affinity enrichment and SILAC for proteomic identification of small molecule-protein interactions. Selective interactions are easily discernable from nonspecific protein binding by quantitative ratios. Using kinase inhibitors as an example, we provide an optimized protocol featuring on-bead protein digestion and single nano-flow liquid chromatographic-mass spectrometric (LC-MS) analyses, consequently increasing analytical throughput and sensitivity over gel-based sample preparation methods for rapid profiling of kinase inhibitor targets.

Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells.

Specific functions for different cyclic nucleotide phosphodiesterases (PDEs) have not yet been identified in most cell types. Conventional approaches to study PDE function typically rely on measurements of global cAMP, general increases in cAMP-dependent protein kinase (PKA), or the activity of exchange protein activated by cAMP (EPAC). Although newer approaches using subcellularly targeted FRET reporter sensors have helped define more compartmentalized regulation of cAMP, PKA, and EPAC, they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem, we have begun to use an unbiased mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the functional pools of cAMP/PKA/EPAC that are regulated by specific cAMP-PDEs (the PDE-regulated phosphoproteomes). In Jurkat cells we find multiple, distinct PDE-regulated phosphoproteomes that can be defined by their responses to different PDE inhibitors. We also find that little phosphorylation occurs unless at least two different PDEs are concurrently inhibited in these cells. Moreover, bioinformatics analyses of these phosphoproteomes provide insight into the unique functional roles, mechanisms of action, and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. The data strongly suggest that the phosphorylation of many different substrates contributes to cAMP-dependent regulation of these cells. The findings further suggest that the approach of using selective, inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling.

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors.

ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile ∼200 kinases in single analytical runs using as little as 5 µL of kinobeads and 300 µg of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2, and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.

SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP.

Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP-SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP/SREBP activation and subsequent regulation of steroidogenesis.