Hiroyuki Inuzuka

SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction.

The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia (T-ALL). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL, validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun, Myc, cyclin E and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth, overexpression of Jun, Myc or notch 1 can also induce programmed cell death. Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCFFBW7 (a SKP1–cullin-1–F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL.

R-cadherin: A novel Ca2+-dependent cell-cell adhesion molecule expressed in the retina

cDNAs encoding a novel member of the cadherin cell adhesion receptor family were cloned. This cadherin is expressed in the retina of the chicken and is termed R-cadherin. It is similar to other cadherins in its primary structure, but most resembles N-cadherin, showing 74% amino acid identity. Cells expressing R-cadherin can adhere to those expressing N-cadherin when mixed, but they form homotypic clusters within their chimeric aggregates. In the development of the neural retina, R-cadherin begins to be expressed around embryonic day 8 in both neuronal and glial cells, and this expression continues up to the hatching stage. The pattern of the expression of R-cadherin was different from that of N-cadherin, suggesting distinctive roles in retinal morphogenesis.

Phosphorylation by Akt1 promotes cytoplasmic localization of Skp2 and impairs APCCdh1-mediated Skp2 destruction.

Deregulated Skp2 function promotes cell transformation, and this is consistent with observations of Skp2 overexpression in many human cancers. However, the mechanisms underlying elevated Skp2 expression are still unknown. Here we show that the serine/threonine protein kinase Akt1, but not Akt2, directly controls Skp2 stability by a mechanism that involves degradation by the APC–Cdh1 ubiquitin ligase complex. We show further that Akt1 phosphorylates Skp2 at Ser 72, which is required to disrupt the interaction between Cdh1 and Skp2. In addition, we show that Ser 72 is localized within a putative nuclear localization sequence and that phosphorylation of Ser 72 by Akt leads to cytoplasmic translocation of Skp2. This finding expands our knowledge of how specific signalling kinase cascades influence proteolysis governed by APC–Cdh1 complexes, and provides evidence that elevated Akt activity and cytoplasmic Skp2 expression may be causative for cancer progression.

mTOR drives its own activation via SCF(βTrCP)-dependent degradation of the mTOR inhibitor DEPTOR.

The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCF(βTrCP). DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds βTrCP. Failure to degrade DEPTOR through either degron mutation or βTrCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.

Roles of F-box proteins in cancer.

F-box proteins, which are the substrate-recognition subunits of SKP1–cullin 1–F-box protein (SCF) E3 ligase complexes, have pivotal roles in multiple cellular processes through ubiquitylation and subsequent degradation of target proteins. Dysregulation of F-box protein-mediated proteolysis leads to human malignancies. Notably, inhibitors that target F-box proteins have shown promising therapeutic potential, urging us to review the current understanding of how F-box proteins contribute to tumorigenesis. As the physiological functions for many of the 69 putative F-box proteins remain elusive, additional genetic and mechanistic studies will help to define the role of each F-box protein in tumorigenesis, thereby paving the road for the rational design of F-box protein-targeted anticancer therapies.

Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus

Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers, and is closely associated with poor prognosis and chemo- or radiotherapeutic resistance. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer.

Phosphorylation by Casein Kinase I promotes the turnover of the Mdm2 oncoprotein via the SCFβ-TRCP ubiquitin ligase

Mdm2 is the major negative regulator of the p53 pathway. Here, we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by casein kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCF(beta-TRCP). Inactivation of either beta-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging agents. Moreover, SCF(beta-TRCP)-dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression.

Restricted expression of N- and R-cadherin on neurites of the developing chicken CNS

The expression of two cadherins, N- and R-cadherin, was mapped in the CNS of chicken embryos of 6–11 d incubation, focusing on the sensory and motor fiber systems. In the spinal cord, the laterally located fibers of the dorsal funiculus express N-cadherin while the medially located fibers do not. These two fiber systems have a different course within the CNS but associate to form the spinal dorsal roots. In the hindbrain, N-cadherin is expressed by the descending trigeminal (general somatic sensory) tract, which is contiguous with the N- cadherin-positive zone of the dorsal funiculus of the spinal cord. R- cadherin is not expressed by sensory fibers, but is expressed by the visceral motor system of the vagus and glossopharyngeal nerves, which are N-cadherin negative. The motor neurites expressing R-cadherin have a different course within the brain than the sensory neurites expressing N-cadherin, although they form the common sensory/motor roots of the vagus nerve at the surface of the brain. The possibility that N-cadherin provides a guidance cue for sensory axon migration within the CNS by a homophilic adhesion mechanism was investigated in vitro. Explants from sensory spinal ganglia expressing N-cadherin were placed on N-cadherin-transfected neuroblastoma cells, and axon outgrowth was visualized. Results showed that the sensory axons defasciculate and closely follow the cell-cell boundaries between transfected cells where high levels of N-cadherin are expressed. These results show that the two cadherins, like members of the immunoglobulin superfamily of molecules, are expressed in a topographically restricted fashion during chick brain development. They furthermore suggest that N- cadherin expression by neurites may play a role in guiding these neurites along CNS paths that express the same molecule.

Proteomic Analysis of β1-Adrenergic Receptor Interactions with PDZ Scaffold Proteins

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of β1-adrenergic receptor (β AR) interactions with PDZ scaffolds, the β1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed β1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel β1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The β1AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of β1AR being the key determinants of the interaction. Full-length β1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the β1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with β1AR profoundly impaired β1AR-mediated ERK1/2 activation but had no apparent effect on β1AR-mediated cyclic AMP generation or agonist-promoted β1AR internalization. These findings revealed that the interaction of MAGI-3 with β1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of β1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which β1 AR signaling and trafficking can be regulated in a cell-specific manner.

Tumor Suppressor Functions of FBW7 in Cancer Development and Progression

FBW7 (F‐box and WD repeat domain‐containing 7) has been characterized as an onco‐suppressor protein in human cancers. Recent studies have also shown that FBW7 exerts its anti‐tumor function primarily by promoting the degradation of various oncoproteins, through which FBW7 regulates cellular proliferation, differentiation and causes genetic instability. In this review, we will discuss the role of FBW7 downstream substrates and how dysregulation of Fbw7‐mediated proteolysis of these substrates contributes to tumorigenesis. Additionally, we will also summarize the currently available various Fbw7‐knockout mouse models that support Fbw7 as a tumor suppressor gene in the development and progression of human malignancies.