Kazumasa Yokoyama

BANK regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP3 receptor

B‐cell activation mediated through the antigen receptor is dependent on activation of protein tyrosine kinases (PTKs) such as Lyn and Syk and subsequent phosphorylation of various signaling proteins. Here we report on the identification and characterization of the B‐cell scaffold protein with ankyrin repeats (BANK), a novel substrate of tyrosine kinases. BANK is expressed in B cells and is tyrosine phosphorylated upon B‐cell antigen receptor (BCR) stimulation, which is mediated predominantly by Syk. Overexpres sion of BANK in B cells leads to enhancement of BCR‐induced calcium mobilization. We found that both Lyn and inositol 1,4,5‐trisphosphate receptor (IP3R) associate with the distinct regions of BANK and that BANK promotes Lyn‐mediated tyrosine phosphorylation of IP3R. Given that IP3R channel activity is up‐regulated by its tyrosine phosphorylation, BANK appears to be a novel scaffold protein regulating BCR‐induced calcium mobilization by connecting PTKs to IP3R. Because BANK expression is confined to functional BCR‐expressing B cells, BANK‐mediated calcium mobilization may be specific to foreign antigen‐induced immune responses rather than to signaling required for B‐cell development.

Depletion of Mammalian CCR4b Deadenylase Triggers Elevation of the p27Kip1 mRNA Level and Impairs Cell Growth

ABSTRACT The stability of mRNA influences the abundance of cellular transcripts and proteins. Deadenylases play critical roles in mRNA turnover and thus are important for the regulation of various biological events. Here, we report the identification and characterization of CCR4b/CNOT6L, which is homologous to yeast CCR4 mRNA deadenylase. CCR4b is localized mainly in the cytoplasm and displays deadenylase activity both in vitro and in vivo. CCR4b forms a multisubunit complex similar to the yeast CCR4-NOT complex. Suppression of CCR4b by RNA interference results in growth retardation of NIH 3T3 cells accompanied by elevation of both p27Kip1 mRNA and p27Kip1 protein. Reintroduction of wild-type CCR4b, but not mutant CCR4b lacking deadenylase activity, restores the growth of CCR4b-depleted NIH 3T3 cells. The data suggest that CCR4b regulates cell growth in a manner dependent on its deadenylase activity. We also show that p27Kip1 mRNA is stabilized and its poly(A) tail is preserved in CCR4b-depleted cells. Our findings provide evidence that CCR4b deadenylase is a constituent of the mammalian CCR4-NOT complex and regulates the turnover rate of specific target mRNAs. Thus, CCR4b may be involved in various cellular events that include cell proliferation.

Involvement of NMDAR2A tyrosine phosphorylation in depression‐related behaviour

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression‐related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression‐related behaviour. The NR2A subunit of the NMDA receptor is tyrosine‐phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr‐1325‐Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock‐in mice show antidepressant‐like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock‐in mice, DARPP‐32 phosphorylation at Thr 34, which is important for the regulation of depression‐related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src‐induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor‐mediated downstream signalling to modulate depression‐related behaviour.

p250GAP, a neural RhoGAP protein, is associated with and phosphorylated by Fyn.

Fyn is a member of the Src-family protein tyrosine kinases and plays important roles in both neurons and oligodendrocytes. Here we report association of Fyn with p250GAP, a RhoGAP protein that is expressed predominantly in brain. p250GAP interacts with Fyn both in vitro and in vivo. p250GAP is tyrosine phosphorylated by Fyn when co-expressed in HEK293T cells. This phosphorylation appears to enhance the interaction between p250GAP and Fyn. Furthermore, the level of tyrosine phosphorylation of p250GAP increases upon differentiation of the oligodendrocyte cell line CG4. Given that Fyn activity is up-regulated during oligodendrocyte maturation, the results argue that p250GAP is phosphorylated by Fyn in oligodendrocytes. Tyrosine phosphorylation of p250GAP by Fyn would regulate its RhoGAP activity, subcellular localization, or interactions with other proteins, leading to morphological and phenotypic changes of oligodendrocytes.

CNOT2 depletion disrupts and inhibits the CCR4–NOT deadenylase complex and induces apoptotic cell death

Eukaryotic mRNA decay is initiated by shortening of the poly (A) tail; however, neither the molecular mechanisms underlying deadenylation nor its regulation is well understood. The human CCR4–NOT complex is a major cytoplasmic deadenylase consisting of a combination of at least nine subunits, four of which have deadenylase activity. The roles of the other subunits remain obscure. Here, we show that CNOT2 depletion by siRNA induces apoptosis. We also show that CNOT2 depletion destabilizes the complex, resulting in the formation of a complex smaller than that formed in control siRNA‐treated cells. The deadenylase activity of the CNOT6L subunit‐containing complex prepared from CNOT2‐depleted cells was less than that from control cells. Intriguingly, the formation of P‐bodies, where mRNA degradation supposedly takes place, was largely suppressed in CNOT2‐depleted cells. Furthermore, CNOT2 depletion enhanced CHOP mRNA levels, suggesting that endoplasmic reticulum (ER) stress was occurring, which causes apoptosis in a caspase‐dependent manner. These results suggest that CNOT2 is important for controlling cell viability through the maintenance of the structural integrity and enzymatic activity of the CCR4–NOT complex.

Focal adhesion kinase regulates laminin-induced oligodendroglial process outgrowth

In the central nervous system (CNS), myelination of axons occurs when oligodendrocyte progenitor cells undergo terminal differentiation, and initiate process formation and axonal ensheathment. Although Fyn, a member of the Src‐family kinases (SFKs), plays an important role in this differentiation process, the substrates of Fyn in oligodendrocytes are largely unknown. Using mass spectrometric analysis, we identified focal adhesion kinase (FAK) as a tyrosine‐phosphorylated protein in the rat‐derived CG4 oligodendrocyte cell line. Tyrosine phosphorylation of FAK was enhanced during differentiation of CG4 cells in a Fyn‐dependent manner. In addition, phosphorylation of FAK was stimulated by laminin, one of the ligands for integrin. Knockdown of FAK expression in CG4 cells suppressed process outgrowth on laminin. Rac1 and Cdc42 activities, which are required for oligodendrocyte process formation, were down‐regulated in FAK‐knockdown cells. Expression of wild‐type (WT) FAK in FAK‐knockdown CG4 cells restored outgrowth of processes, but the Y397F mutant lacking the autophosphorylation site did not. These results suggest that FAK/Fyn‐mediated activation of Rac1 and Cdc42 is critical for laminin‐induced outgrowth of oligodendrocyte processes.

Distinct expression patterns of the subunits of the CCR4-NOT deadenylase complex during neural development.

The stability of mRNA influences the dynamics of gene expression. The mammalian CCR4-NOT complex is associated with deadenylase activity, which shortens the mRNA poly(A) tail and thereby contributes to destabilization of mRNAs. The complex consists of at least nine subunits and predominantly forms a 2.0MDa protein complex in HeLa cells. Accumulating evidence suggests that the CCR4-NOT complex is involved in cell growth and survival; however, the regulatory mechanisms of its biological activity remain obscure. Here, we analyzed the expression levels of the subunits of the CCR4-NOT complex in various mouse tissues and found that they showed distinct expression patterns. CNOT6, 6L, 7, and 10 were expressed nearly ubiquitously, whereas others were expressed in tissue-specific manners, such as those displaying especially high expression in the brain. Furthermore, CNOT2, 3, 6, and 8 were rapidly downregulated during differentiation of neural stem cells. These findings suggest that subunit composition of the CCR4-NOT complex differs among tissues and is altered during neural development, thereby imparting an additional layer of specificity in the control of gene expression.

Azoospermia in mice with targeted disruption of the Brek/Lmtk2 (brain-enriched kinase/lemur tyrosine kinase 2) gene

Brek/Lmtk2 (brain-enriched kinase/lemur tyrosine kinase 2) is a member of the Aatyk family of kinases that comprises Aatyk1, Brek/Lmtk2/Aatyk2, and Aatyk3. Although several potential roles have been proposed for Brek and other Aatyk family members, the physiological functions of these kinases remain unclear. Here, we report that Brek−/− male mice are infertile, with azoospermia. Detailed histological analysis revealed that Brek−/− germ cells differentiated normally until the round-spermatid stage, but failed to undergo the normal change in morphology to become elongated spermatids. Testicular somatic cells appeared normal in these mice. Expression of Brek in testis was restricted to the germ cells, suggesting that the maturations of germ cells in Brek−/− mice are affected in a cell-autonomous manner. On the basis of these findings, we concluded that Brek is essential for a late stage of spermatogenesis. Further clarification of the mechanism by which Brek regulates spermatogenesis may help identify new targets for reproductive contraceptives and treatments against infertility.

NYAP: a phosphoprotein family that links PI3K to WAVE1 signalling in neurons

The phosphoinositide 3‐kinase (PI3K) pathway has been extensively studied in neuronal function and morphogenesis. However, the precise molecular mechanisms of PI3K activation and its downstream signalling in neurons remain elusive. Here, we report the identification of the Neuronal tYrosine‐phosphorylated Adaptor for the PI 3‐kinase (NYAP) family of phosphoproteins, which is composed of NYAP1, NYAP2, and Myosin16/NYAP3. The NYAPs are expressed predominantly in developing neurons. Upon stimulation with Contactin5, the NYAPs are tyrosine phosphorylated by Fyn. Phosphorylated NYAPs interact with PI3K p85 and activate PI3K, Akt, and Rac1. Moreover, the NYAPs interact with the WAVE1 complex which mediates remodelling of the actin cytoskeleton after activation by PI3K‐produced PIP3 and Rac1. By simultaneously interacting with PI3K and the WAVE1 complex, the NYAPs bridge a PI3K–WAVE1 association. Disruption of the NYAP genes in mice affects brain size and neurite elongation. In conclusion, the NYAPs activate PI3K and concomitantly recruit the downstream effector WAVE complex to the close vicinity of PI3K and regulate neuronal morphogenesis.

NMDAR2B tyrosine phosphorylation regulates anxiety-like behavior and CRF expression in the amygdala

BackgroundAnxiety disorders are a highly prevalent and disabling class of psychiatric disorders. There is growing evidence implicating the glutamate system in the pathophysiology and treatment of anxiety disorders, though the molecular mechanism by which the glutamate system regulates anxiety-like behavior remains unclear.ResultsIn this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to anxiety-like behavior. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated: Tyr-1472 is the major phosphorylation site. Homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B, which prevents phosphorylation of this site, show enhanced anxiety-like behavior in the elevated plus-maze test. Expression of corticotropin-releasing factor (CRF), which is important for the regulation of anxiety-like behavior, is increased in the amygdala of the knock-in mice. Furthermore, injection of CRF receptor antagonist attenuated the enhanced anxiety-like behavior of the knock-in mice. We also show that elevated plus-maze exposure simultaneously induced de-phosphorylation of Tyr-1472 and increased CRF expression.ConclusionsThese data suggest that Tyr-1472 phosphorylation on GluN2B is important for anxiety-like behavior by negative regulation of CRF expression in the amygdala.