Beta-amyloid interacts with and activates the long-form phosphodiesterase PDE4D5 in neuronal cells to reduce cAMP availability.

Inhibition of the cyclic-AMP degrading enzyme phosphodiesterase type 4 (PDE4) in the brains of animal models is protective in Alzheimer's disease (AD). We show for the first time that enzymes from the subfamily PDE4D not only colocalize with beta-amyloid (Aß) plaques in a mouse model of AD but that Aß directly associates with the catalytic machinery of the enzyme. Peptide mapping suggests that PDE4D is the preferential PDE4 subfamily for Aß as it possesses a unique binding site. Intriguingly, exogenous addition of Aß to cells overexpressing the PDE4D5 longform caused PDE4 activation and a decrease in cAMP. We suggest a novel mechanism where PDE4 longforms can be activated by Aß, resulting in the attenuation of cAMP signalling to promote loss of cognitive function in AD.

Disruption of the pro-oncogenic c-RAF-PDE8A complex represents a differentiated approach to treating KRAS-c-RAF dependent PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF-PDE8A protein-protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF-cAMP/PKA signaling axes and treating KRAS-c-RAF dependent PDAC. Through disrupting the c-RAF-PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS-c-RAF dependent PDAC.

cAMP-phosphodiesterase 4D7 (PDE4D7) forms a cAMP signalosome complex with DHX9 and is implicated in prostate cancer progression.

A robust body of work has demonstrated that a reduction in cAMP-specific 3',5'-cyclic phosphodiesterase 4D isoform 7 (PDE4D7) is linked with negative prostate cancer outcomes; however, the exact molecular mechanism that underpins this relationship is unknown. Epigenetic profiling has shown that the PDE4D gene can be hyper-methylated in transmembrane serine protease 2 (TMPRSS2)-ETS transcriptional regulator ERG (ERG) gene-fusion-positive prostate cancer (PCa) tumours, and this inhibits messenger RNA (mRNA) expression, leading to a paucity of cellular PDE4D7 protein. In an attempt to understand how the resulting aberrant cAMP signalling drives PCa growth, we immunopurified PDE4D7 and identified binding proteins by mass spectrometry. We used peptide array technology and proximity ligation assay to confirm binding between PDE4D7 and ATP-dependent RNA helicase A (DHX9), and in the design of a novel cell-permeable disruptor peptide that mimics the DHX9-binding region on PDE4D7. We discovered that PDE4D7 forms a signalling complex with the DExD/H-box RNA helicase DHX9. Importantly, disruption of the PDE4D7-DHX9 complex reduced proliferation of LNCaP cells, suggesting the complex is pro-tumorigenic. Additionally, we have identified a novel protein kinase A (PKA) phosphorylation site on DHX9 that is regulated by PDE4D7 association. In summary, we report the existence of a newly identified PDE4D7-DHX9 signalling complex that may be crucial in PCa pathogenesis and could represent a potential therapeutic target.

Revisiting the roles of cAMP signalling in the progression of prostate cancer.

Prostate cancer is one of the most common cancers in men and one of the top causes of death in men worldwide. Development and function of both normal prostate cells and early-stage prostate cancer cells are dependent on the cross-talk between androgen signalling systems and a variety of other transduction pathways which drive differentiation of these cells towards castration-resistance. One such signalling pathway is the ubiquitous cAMP signalling axis which functions to activate spatially restricted pools of cAMP effectors such as protein kinase A (PKA). The importance of both PKA and cAMP in the development of prostate cancer, and their interactions with the androgen receptor, were the focus of a review by Merkle and Hoffmann in 2010. In this updated review, we revisit this topic with analysis of current PKA-related prostate cancer literature and introduce novel information on the relevance of another cAMP effector, the exchange protein directly activated by cAMP (EPAC).

Reduced PDE4D7 in prostate cancer correlates with genomic downregulation within the upstream PDE4D coding region.

Aim: PDE4D7 expression is significantly associated with prostate cancer (PCa) progression, representing an attractive prognostic biomarker. We sought to determine whether other genes in the PDE4D coding region were associated. Patients & methods: RNA from biopsy punch samples of resected tumor tissue was analyzed via RNA sequencing. RT-qPCR was used to determine PDE4D7 score. Results: Numerous genomic sequences within the PDE4D coding region on Chr5q12 revealed similar mRNA expression profiles to PDE4D7. PART1 had a significantly similar expression pattern to PDE4D7 across samples, correlating with disease progression. However, many other genes also exhibited matched expression to PDE4D7, including miRNAs and lncRNAs. Conclusion: These novel PDE4D7-associated genes, many of which are previously uncharacterized in cancer, represent putative PCa biomarkers and could have mechanistic roles in PCa progression.

Identification of biological molecules that can indicate the presence of a disease are known as biomarkers and are valuable in medical research. For prostate cancer, the PDE4D7 gene is helpful for determining how the disease is getting worse. To understand more, this study explored whether genes located near PDE4D7 are also connected to prostate cancer. The analysis of tumor biopsies revealed that many of these nearby genes exhibited similar expression levels to PDE4D7, indicating their association with disease development. These findings suggest that several of these nearby genes could serve as potential biomarkers for prostate cancer by giving important clues to understand and detect the disease better.

Loss of PDE4D7 expression promotes androgen independence, neuroendocrine differentiation and alterations in DNA repair: implications for therapeutic strategies.

BACKGROUND: Androgen signalling remains the seminal therapeutic approach for the management of advanced prostate cancer. However, most tumours eventually shift towards an aggressive phenotype, characterised by androgen independence and treatment resistance. The cyclic adenosine monophosphate (cAMP) pathway plays a crucial role in regulating various cellular processes, with the phosphodiesterase PDE4D7 being a vital modulator of cAMP signalling in prostate cancer cells. METHODS: Using shRNA-mediated PDE4D7 knockdown in LNCaP cells and downstream analysis via RNA sequencing and phenotypic assays, we replicate clinical observations that diminished PDE4D7 expression promotes an aggressive prostate cancer phenotype. RESULTS: Our study provides evidence that loss of PDE4D7 expression represents a pivotal switch driving the transition from an androgen-sensitive state to hormone unresponsiveness and neuroendocrine differentiation. In addition, we demonstrate that PDE4D7 loss affects DNA repair pathways, conferring resistance to poly ADP ribose polymerase (PARP) inhibitors. CONCLUSION: Reinstating PDE4D7 expression sensitises prostate cancer cells to anti-androgens, DNA damage response inhibitors, and cytotoxic therapies. These findings provide significant insight into the regulatory role of PDE4D7 in the development of lethal prostate cancer and the potential of its modulation as a novel therapeutic strategy.

Short PDE4 Isoforms as Drug Targets in Disease.

The second messenger, cyclic adenosine monophosphate (cAMP), is a master regulator of signal transduction that maintains cell homeostasis. A fine balance between cAMP synthesis by adenylyl cyclase and degradation by phosphodiesterases (PDEs) underpins receptor-specific responses. As multiple receptors rely on cAMP for signaling, PDEs shape three-dimensional, localized gradients of the cyclic nucleotide to drive appropriate signaling cascades. Of the 11 PDE families, PDE4, which comprises long, short, and supershort isoforms and a dead-short isoform, is of great interest due to its implication in disease. Aberrant PDE4 expression and post-translational modifications are hallmarks of several clinical indications for which curative treatment is not yet available. While some PDE4-specific small molecule inhibitors directed against the active site are approved for clinical use, they are limited by severe side effects owing to the high degree of conservation of the catalytic domain between over 20 unique isoforms. Some attempts to use the different modular structure that exists between long and shorter isoforms are now bearing success. However, these inhibitors are exclusively aimed at PDE4 long isoforms, which have been the focus of the majority of research in this area. Here, we have summarised literature on the lesser-studied short PDE4 isoforms and provide a record of the discovery, regulation, and disease relevance of this class of enzymes that represent an untapped target for specific inhibition in the future.

Cancer: Phosphodiesterase type 4C (PDE4C), the forgotten subfamily as a therapeutic target.

Phosphodiesterase type 4 (PDE4) enzymes specifically hydrolyse cAMP in many cell signalling systems that are transduced by hormones and other primary messengers. The physiological function of the four PDE4 subfamilies (A, B, C and D) are numerous and varied due to the differentially localised plethora of isoforms that can be detected in cardiovascular, CNS and immune systems. Of the four subfamilies, least is known about PDE4C probably due to its restricted distribution pattern, scarcity of selective inhibitors and the lack of developed research tools. Here, for the first time, we chart the discovery of PDE4C, describe its regulation and highlight cancers where future development of PDE4C selective small molecules may have potential.

Ablation of specific long PDE4D isoforms increases neurite elongation and conveys protection against amyloid-ß pathology.

Inhibition of phosphodiesterase 4D (PDE4D) enzymes has been investigated as therapeutic strategy to treat memory problems in Alzheimer's disease (AD). Although PDE4D inhibitors are effective in enhancing memory processes in rodents and humans, severe side effects may hamper their clinical use. PDE4D enzymes comprise different isoforms, which, when targeted specifically, can increase treatment efficacy and safety. The function of PDE4D isoforms in AD and in molecular memory processes per se has remained unresolved. Here, we report the upregulation of specific PDE4D isoforms in transgenic AD mice and hippocampal neurons exposed to amyloid-ß. Furthermore, by means of pharmacological inhibition and CRISPR-Cas9 knockdown, we show that the long-form PDE4D3, -D5, -D7, and -D9 isoforms regulate neuronal plasticity and convey resilience against amyloid-ß in vitro. These results indicate that isoform-specific, next to non-selective, PDE4D inhibition is efficient in promoting neuroplasticity in an AD context. Therapeutic effects of non-selective PDE4D inhibitors are likely achieved through actions on long isoforms. Future research should identify which long PDE4D isoforms should be specifically targeted in vivo to both improve treatment efficacy and reduce side effects.

Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor.

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the progression and invasiveness of melanoma. MITF-regulated gene expression requires recruitment of the transcriptional co-regulator CBP/p300, but details of this process are not fully defined. In this study, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. Using pulldown assays and nuclear magnetic resonance spectroscopy we determined that MITFTAD is intrinsically disordered and binds to the TAZ1 and TAZ2 domains of CBP/p300 with moderate affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining somewhat dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These findings provide insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies.

S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodium-calcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Little is known about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression increased rapidly during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca2.

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.

Ataxia-telangiectasia mutated and ataxia telangiectasia and Rad3-related kinases as therapeutic targets and stratification indicators for prostate cancer.

The DNA damage response is an integral part of a cells' ability to maintain genomic integrity by responding to and ameliorating DNA damage, or initiating cell death for irrepairably damaged cells. This response is often hijacked by cancer cells to evade cell death allowing mutant cells to persist, as well as in the development of treatment resistance to DNA damaging agents such as chemotherapy and radiation. Prostate cancer (PCa) cells often exhibit alterations in DNA damage response genes including ataxia telangiectasia mutated (ATM), correlating with aggressive disease phenotype. The recent success of Poly (ADP-ribose) polymerase (PARP) inhibition has led to several clinically approved PARP inhibitors for the treatment of men with metastatic PCa, however a key limitation is the development of drug resistance and relapse. An alternative approach is selectively targeting ATM and ataxia telangiectasia and Rad3-related (ATR) which, due to their position at the forefront of the DDR, represent attractive pharmacological targets. ATR inhibition has been shown to act synergistically with PARP inhibition and other cancer treatments to enhance anti-tumour activity. ATM-deficiency is a common characteristic of PCa and a synthetic lethal relationship exists between ATM and ATR, with ATR inhibition inducing selective cell death in ATM-deficient PCa cells. The current research highlights the feasibility of therapeutically targeting ATR in ATM-deficient prostate tumours and in combination with other treatments to enhance overall efficacy and reduce therapeutic resistance. ATM also represents an important molecular biomarker to stratify patients into targeted treatment groups and aid prognosis for personalised medicine.

CMYA5 is a novel interaction partner of FHL2 in cardiac myocytes.

Four-and-a-half LIM domains protein 2 (FHL2) is an anti-hypertrophic adaptor protein that regulates cardiac myocyte signalling and function. Herein, we identified cardiomyopathy-associated 5 (CMYA5) as a novel FHL2 interaction partner in cardiac myocytes. In vitro pull-down assays demonstrated interaction between FHL2 and the N- and C-terminal regions of CMYA5. The interaction was verified in adult cardiac myocytes by proximity ligation assays. Immunofluorescence and confocal microscopy demonstrated co-localisation in the same subcellular compartment. The binding interface between FHL2 and CMYA5 was mapped by peptide arrays. Exposure of neonatal rat ventricular myocytes to a CMYA5 peptide covering one of the FHL2 interaction sites led to an increase in cell area at baseline, but a blunted response to chronic phenylephrine treatment. In contrast to wild-type hearts, loss or reduced FHL2 expression in Fhl2-targeted knockout mouse hearts or in a humanised mouse model of hypertrophic cardiomyopathy led to redistribution of CMYA5 into the perinuclear and intercalated disc region. Taken together, our results indicate a direct interaction of the two adaptor proteins FHL2 and CMYA5 in cardiac myocytes, which might impact subcellular compartmentation of CMYA5.

Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.

The CAPRA&PDE4D5/7/9 Prognostic Model Is Significantly Associated with Adverse Post-Surgical Pathology Outcomes.

Objectives: To investigate the association of the prognostic risk score CAPRA&PDE4D5/7/9 as measured on pre-surgical diagnostic needle biopsy tissue with pathological outcomes after radical prostatectomies in a clinically low−intermediate-risk patient cohort. Patients and Methods: RNA was extracted from biopsy punches of diagnostic needle biopsies. The patient cohort comprises n = 151 patients; of those n = 84 had low−intermediate clinical risk based on the CAPRA score and DRE clinical stage 2, or pathological pT stage > pT3a, or tumor penetrated prostate capsular status, or pN1 disease); (ii) any ISUP pathological Gleason >2; (iii) any ISUP pathological Gleason >1. In the n = 84 patients with low to intermediate clinical risk profiles, the clinical-genomics CAPRA&PDE4D5/7/9_BCR risk score was significantly lower in patients with favorable vs. unfavorable outcomes. In univariable logistic regression modeling the genomics PDE4D5/7/9_BCR as well as the clinical-genomics CAPRA&PDE4D5/7/9_BCR combination model were significantly associated with all three post-surgical pathology outcomes (p = 0.02, p = 0.0004, p = 0.04; and p = 0.01, p = 0.0002, p = 0.01, respectively). The clinically used PRIAS criteria for the selection of low-risk candidate patients for active surveillance (AS) were not significantly associated with any of the three tested post-operative pathology outcomes (p = 0.3, p = 0.1, p = 0.1, respectively). In multivariable analysis adjusted for the CAPRA score, the genomics PDE4D5/7/9_BCR risk score remained significant for the outcomes of adverse pathology (p = 0.04) and ISUP pathological Gleason >2 (p = 0.004). The negative predictive value of the CAPRA&PDE4D5/7/9_BCR risk score using the low-risk cut-off (0.1) for the three pathological endpoints was 82.0%, 100%, and 59.1%, respectively for a selected low-risk cohort of n = 22 patients (26.2% of the entire cohort) compared to 72.1%, 94.4%, and 55.6% for n = 18 low-risk patients (21.4% of the total cohort) selected based on the PRIAS inclusion criteria. Conclusion: In this study, we have shown that the previously reported clinical-genomics prostate cancer risk model CAPRA&PDE4D5/7/9_BCR which was developed to predict biological outcomes after surgery of primary prostate cancer is also significantly associated with post-surgical pathology outcomes. The risk score predicts adverse pathology independent of the clinical risk metrics. Compared to clinically used active surveillance inclusion criteria, the clinical-genomics CAPRA&PDE4D5/7/9_BCR risk model selects 22% (n = 8) more low-risk patients with higher negative predictive value to experience unfavorable post-operative pathology outcomes.

Peptides derived from the SARS-CoV-2 receptor binding motif bind to ACE2 but do not block ACE2-mediated host cell entry or pro-inflammatory cytokine induction.

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)-ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low µM range (Kd = 0.207-1.206 µM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, 'miniproteins', nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides.

Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149-811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

The RanBP2/RanGAP1-SUMO complex gates ß-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein ß-arrestin2 (ß-arr2) regulates tumor suppressor p53 by counteracting Mdm2. ß-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. ß-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. ß-arr2 can be SUMOylated, but no information is available on how SUMO may regulate ß-arr2 nucleocytoplasmic shuttling. While we found ß-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and ß-arr2, via a SUMO interaction motif (SIM), that is required for ß-arr2 cytonuclear trafficking. This SIM promotes association of ß-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective ß-arr2 nuclear entry. Mutation of the SIM inhibits ß-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a ß-arr2 SIM nuclear entry checkpoint, coupled with active ß-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.