Futoshi Suizu

Regulation of NF-κB Signaling by Pin1-Dependent Prolyl Isomerization and Ubiquitin-Mediated Proteolysis of p65/RelA

The transcription factor NF-kappaB is activated by the degradation of its inhibitor IkappaBalpha, resulting in its nuclear translocation. However, the mechanism by which nuclear NF-kappaB is subsequently regulated is not clear. Here we demonstrate that NF-kappaB function is regulated by Pin1-mediated prolyl isomerization and ubiquitin-mediated proteolysis of its p65/RelA subunit. Upon cytokine treatment, Pin1 binds to the pThr254-Pro motif in p65 and inhibits p65 binding to IkappaBalpha, resulting in increased nuclear accumulation and protein stability of p65 and enhanced NF-kappaB activity. Significantly, Pin1-deficient mice and cells are refractory to NF-kappaB activation by cytokine signals. Moreover, the stability of p65 is controlled by ubiquitin-mediated proteolysis, facilitated by a cytokine signal inhibitor, SOCS-1, acting as a ubiquitin ligase. These findings uncover two important mechanisms of regulating NF-kappaB signaling and offer new insight into the pathogenesis and treatment of some human diseases such as cancers.

Phosphorylation-specific prolyl isomerization: is there an underlying theme?

The prolyl isomerase Pin1 is a conserved enzyme that is intimately involved in diverse biological processes and pathological conditions such as cancer and Alzheimers disease. By catalysing cis–trans interconversion of certain motifs containing phosphorylated serine or threonine residues followed by a proline residue (pSer/Thr-Pro), Pin1 can have profound effects on phosphorylation signalling. The structural and functional differences that result from cis–trans isomerization of specific pSer/Thr-Pro motifs probably underlie most, if not all, Pin1-dependent actions. Phosphorylation-dependent prolyl isomerization by Pin1 remains a unique mode for the modulation of signal transduction. Here, we provide an overview of the plethora of regulatory events that involve this unique enzyme, with a particular focus on oncogenic signalling and neurodegeneration.

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells.

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper‐interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642–1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin‐activated MgATPase activity of myosin II. HeLa ZIPK is the first non‐muscle MLCK that phosphorylates MRLC at two sites.

Pin1 Regulates Centrosome Duplication, and Its Overexpression Induces Centrosome Amplification, Chromosome Instability, and Oncogenesis

ABSTRACT Phosphorylation on Ser/Thr-Pro motifs is a major mechanism regulating many events involved in cell proliferation and transformation, including centrosome duplication, whose defects have been implicated in oncogenesis. Certain phosphorylated Ser/Thr-Pro motifs can exist in two distinct conformations whose conversion in certain proteins is catalyzed specifically by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and is important for the activation of multiple oncogenic pathways, and its deletion suppresses the ability of certain oncogenes to induce cancer in mice. However, little is known about the role of Pin1 in centrosome duplication and the significance of Pin1 overexpression in cancer development in vivo. Here we show that Pin1 overexpression correlates with centrosome amplification in human breast cancer tissues. Furthermore, Pin1 localizes to and copurifies with centrosomes in interphase but not mitotic cells. Moreover, Pin1 ablation in mouse embryonic fibroblasts drastically delays centrosome duplication without affecting DNA synthesis and Pin1 inhibition also suppresses centrosome amplification in S-arrested CHO cells. In contrast, overexpression of Pin1 drives centrosome duplication and accumulation, resulting in chromosome missegregation, aneuploidy, and transformation in nontransformed NIH 3T3 cells. More importantly, transgenic overexpression of Pin1 in mouse mammary glands also potently induces centrosome amplification, eventually leading to mammary hyperplasia and malignant mammary tumors with overamplified centrosomes. These results demonstrate for the first time that the phosphorylation-specific isomerase Pin1 regulates centrosome duplication and its deregulation can induce centrosome amplification, chromosome instability, and oncogenesis.

The E3 Ligase TTC3 Facilitates Ubiquitination and Degradation of Phosphorylated Akt

The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.

Proto-oncogene TCL1: more than just a coactivator for Akt

Serine threonine kinase Akt, also called PKB (protein kinase B), plays a central role in regulating intracellular survival. Deregulation of this Akt signaling pathway underlies various human neoplastic diseases. Recently, the proto‐oncogene TCL1 (T cell leukemia 1), with a previously unknown physiological function, was shown to interact with the Akt pleckstrin homology domain, enhancing Akt kinase activity; hence, it functions as an Akt kinase coactivator. In contrast to pathological conditions in which the TCL1 gene is highly activated in various human neoplasmic diseases, the physiological expression of TCL1 is tightly limited to early developmental cells as well as various developmental stages of immune cells. The NBRE (nerve growth factor‐responsive element) of the proximal TCL1 promoter sequences can regulate the restricted physiological expression of TCL1 in a negative feedback mechanism. Further, based on the NMR structural studies of Akt‐TCL1 protein complexes, an inhibitory peptide, “Akt‐in,” consisting of the βA strand of TCL1, has been identified and has therapeutic potential. This review article summarizes and discusses recent advances in the understanding of TCL1‐Akt functional interaction in order to clarify the biological action of the proto‐oncogene TCL1 family and the development avenues for a suppressive drug specific for Akt, a core intracellular survival regulator.—Noguchi, M., Ropars, V., Roumestand, C., Suizu, F. Proto‐oncogene TCL1: more than just a coactivator for Akt. FASEB J. 21, 2273–2284 (2007)

Targeting carcinogenesis: a role for the prolyl isomerase Pin1?

Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr‐Pro) is a central signaling mechanism in cell proliferation and transformation. Recent studies indicate that certain pSer/Thr‐Pro motifs in native proteins exist in two completely distinct conformations, cis and trans, whose conversion is markedly slowed down upon phosphorylation, but specifically catalyzed by the peptidyl‐prolyl cis/trans isomerase Pin1. Importantly, such Pin1‐catalyzed conformational changes can have profound effects on the function of many phosphorylation signaling pathways, thereby playing an important role in various cellular processes. Moreover, increasing evidence indicates that aberrant Pin1 function plays an important role in the pathogenesis of some human diseases. Notably, Pin1 is not only overexpressed in a large number of human cancers, but also is an excellent prognostic marker in some cancers. Furthermore, Pin1 overexpression can function as a critical catalyst that amplifies multiple oncogenic signaling pathways during oncogenesis. Moreover, Pin1 overexpression causes cell transformation, centrosome amplification, genomic instability, and tumor development. In contrast, Pin1 knockout in mice prevents certain oncogenes from inducing tumors and Pin1 inhibition in cancer cells suppresses their cell proliferation, transformed phenotype and tumorigenicity in nude mice as well as increases the response to other anticancer agents. These results suggest that Pin1‐mediated postphosphorylation regulation may provide a unique opportunity for disrupting oncogenic pathways, and thereby represent an appealing target for novel anticancer therapies.

Identification of nerve growth factor-responsive element of the TCL1 promoter as a novel negative regulatory element

The serine/threonine kinase, Akt (protein kinase B) plays a central role in the regulation of intracellular cell survival. Recently, we demonstrated that the proto-oncogene TCL1, overexpressed in human T-cell prolymphocytic leukemia, is an Akt kinase co-activator. Tightly restricted TCL1 gene expression in early developmental cells suggested that the TCL1 gene is regulated at a transcriptional level. To characterize how TCL1 gene expression is regulated, we cloned the 5′-promoter of the TCL1 gene located at human chromosome 14q32. The 5′-TCL1 promoter region contains a TATA box with cis-regulatory elements for Nur77/NGFI-B (nerve growth factor-responsive element (NBRE), CCAAGGTCA), NFκB, and fork head transcription factor. Nur77/NGFI-B, an orphan receptor superfamily transcription factor implicated in T-cell apoptosis, is a substrate for Akt. We hypothesized that TCL1 transactivity is regulated through Akt-induced phosphorylation of Nur77/NGFI-B in vivo. In an electrophoretic mobility shift assay with chromosomal immunoprecipitation assays, wild-type Nur77, but not S350A mutant Nur77, could specifically bind to TCL1-NBRE. A luciferase assay demonstrated that TCL1-NBRE is required for inhibition of TCL1 transactivity upon nerve growth factor/platelet-derived growth factor stimulation, which activates Akt and phosphorylates Nur77. Using a chromosomal immunoprecipitation assay with reverse transcription-PCR, nerve growth factor stimulation inhibited binding of endogenous Nur77 to TCL1-NBRE, in turn, suppressing TCL1 gene expression. The results together establish that TCL1-NBRE is a novel negative regulatory element of Nur77 (NGFI-B). To the best of our knowledge, TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death survival. This Akt-induced inhibitory mechanism of TCL1 should play an important role in immunological and/or neuronal development in vivo.

The links between AKT and two intracellular proteolytic cascades: Ubiquitination and autophagy

The serine threonine kinase AKT plays a central role in the regulation of cell survival in a variety of human neoplastic diseases. A series of studies have revealed a connection between AKT signaling and two important protein degradation pathways in mammalian cells: the ubiquitin-proteasome system and autophagy. Two distinct ubiquitination systems have been reported to regulate AKT signaling: K63-linked ubiquitination, which promotes the oncogenic activation of AKT, and K48-linked ubiquitination, which triggers the proteasomal degradation of phosphorylated AKT. Autophagy is an evolutionarily conserved mechanism for the gross disposal and recycling of intracellular proteins in mammalian cells. AKT signaling may play a regulatory role in autophagy; however, the underlying mechanisms have not been fully clarified. Recently, AKT was shown to phosphorylate key molecules involved in the regulation of autophagy. Furthermore, lysosomal co-localization of the AKT-Phafin2 complex is reportedly critical for the induction of autophagy. In this review, we will discuss the connection between AKT, a core intracellular survival regulator, and two major intracellular proteolytic signaling pathways in mammalian cells.

Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo.

Ca(2+)/calmodulin (CaM)-dependent protein kinase I (CaM-KI), which is a member of the multifunctional CaM-K family, is thought to be involved in various Ca(2+)-signalling pathways. In this report, we demonstrate that CaM-KI activated by an upstream kinase (CaM-K kinase), but not unactivated CaM-KI, phosphorylates myosin II regulatory light chain (MRLC) efficiently ( K (cat), 1.7 s(-1)) and stoichiometrically (approximately 0.8 mol of phosphate/mol) in a Ca(2+)/CaM-dependent manner in vitro. One-dimensional phosphopeptide mapping and mutational analysis of MRLC revealed that the activated CaM-KI monophosphorylates only Ser-19 in MRLC. Transient expression of the Ca(2+)/CaM-independent form of CaM-KI (CaM-KI(1-293)) in HeLa cells induced Ser-19 phosphorylation of myosin, II accompanied by reorganization of actin filaments in the peripheral region of the cells. CaM-KI-induced reorganization of actin filaments was suppressed by co-expression of non-phosphorylatable MRLC mutants (S19A and T18AS19A). Furthermore, a kinase-negative form of CaM-KI (CaM-KI(1-293,K49E)) significantly reduced reorganization of actin filaments, indicating a dominant negative effect. This is the first demonstration that the activation of the CaM-KI cascade induces myosin II phosphorylation, resulting in regulation of actin filament organization in mammalian cells.