The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity

p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The pak3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.     

猪PILRA基因克隆及变异剪接体鉴定

Ⅱ型成对免疫球蛋白样受体(Paired immunoglobulin-like type 2 receptors, PILRs)是免疫球蛋白超家族成员之一,包括α和β两个亚型。PILRα在机体抵抗病原体入侵的免疫反应中发挥着重要作用,但目前尚未见关于猪PILRα的报道。为了分析其在猪抗病育种中的作用,本文克隆了猪PILRα编码基因(PILRA)并鉴定变异剪接体,利用实时荧光定量PCR方法构建其组织表达谱和诱导表达谱。结果表明,成功克隆了猪PILRA基因的3个变异剪接体V1~V3(GenBank登录号：KJ143679~81),预测的蛋白质多肽链分别长271 aa、254 aa和283 aa,且都具有免疫球蛋白结构域。各变异剪接体均在脾脏中表达量最高,肝脏、肺脏次之,在心脏、肾脏、胃、肌肉、淋巴、大肠、小肠和膀胱等组织中表达量较低或检测不到。Poly(I:C)能显著诱导变异剪接体V1的表达,但几乎不影响V2、V3的表达。研究结果为进一步揭示PILRA在猪抗病育种中的作用提供了基础。     

Differential expression and developmental regulation of a novel alpha-dystrobrevin isoform in muscle

Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.     

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.     

Expression profile and characterisation of a truncated KCNQ5 splice variant

Ion channels encoded by KCNQ genes (1-5) are key regulators of membrane properties in many cell types. The KCNQ5 gene was the last to be identified and has three splice variants that are expressed in human brain and skeletal muscle. The KCNQ5 encoded channel possesses M-current properties and so far no channelopathy has been associated with any of the three variants. We now show that only the shortest KCNQ5 variant, which has exon 9 deleted, was expressed in a variety of murine vascular smooth muscle. In Xenopus oocytes, this variant generated currents with amplitudes, activation kinetics and biophysical properties similar to the full-length variant normally expressed in neuronal tissue. Furthermore sensitivity to block by XE991 and activation by retigabine were also similar between both variants. These data represent an exhaustive characterisation of a truncated KCNQ5 splice variant that may contribute to the native XE991-sensitive channel in murine vasculature.     

Synaptic Effects of Munc18-1 Alternative Splicing in Excitatory Hippocampal Neurons

The munc18-1 gene encodes two splice-variants that vary at the C-terminus of the protein and are expressed at different levels in different regions of the adult mammalian brain. Here, we investigated the expression pattern of these splice variants within the brainstem and tested whether they are functionally different. Munc18-1a is expressed in specific nuclei of the brainstem including the LRN, VII and SOC, while Munc18-1b expression is relatively low/absent in these regions. Furthermore, Munc18-1a is the major splice variant in the Calyx of Held. Synaptic transmission was analyzed in autaptic hippocampal munc18-1 KO neurons re-expressing either Munc18-1a or Munc18-1b. The two splice variants supported synaptic transmission to a similar extent, but Munc18-1b was slightly more potent in sustaining synchronous release during high frequency stimulation. Our data suggest that alternative splicing of Munc18-1 support synaptic transmission to a similar extent, but could modulate presynaptic short-term plasticity.     

A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.     

SULF1/SULF2 splice variants differentially regulate pancreatic tumour growth progression

This study highlights the highly dynamic nature of SULF1/SULF2 splice variants in different human pancreatic cancers that regulate the activities of multiple cell signalling pathways in development and disease. Most pancreatic tumours expressed variable levels of both SULF1 and SULF2 variants including some expression during inflammation and pancreatitis. Many ductal and centro-acinar cell-derived pancreatic tumours are known to evolve into lethal pancreatic ductal adenocarcinomas but the present study also detected different stages of such tumour progression in the same tissue biopsies of not only acinar cell origin but also islet cell-derived cancers. The examination of caerulein-induced pancreatic injury and tumorigenesis in a Kras-driven mouse model confirmed the activation and gradual increase of SULF1/SULF2 variants during pancreatitis and tumorigenesis but with reduced levels in Stat3 conditional knockout mice with reduced inflammation. The significance of differential spatial and temporal patterns of specific SULF1/SULF2 splice variant expression during cancer growth became further apparent from their differential stimulatory or inhibitory effects on growth factor activities, tumour growth and angiogenesis not only during in vitro but also in vivo growth thus providing possible novel therapeutic targets.     

Unifying nomenclature for the isoforms of the lysosomal membrane protein LAMP-2

The present nomenclature of the splice variants of the lysosome-associated membrane protein type 2 (LAMP-2) is confusing. The LAMP-2a isoform is uniformly named in human, chicken, and mouse, but the LAMP-2b and LAMP-2c isoforms are switched in human as compared with mouse and chicken. We propose to change the nomenclature of the chicken and mouse b and c isoforms to agree with that currently used for the human isoforms. To avoid confusion in the literature, we further propose to adopt the use of capital letters for the updated nomenclature of all the isoforms in all three species: LAMP-2A, LAMP-2B, and LAMP-2C.     

A variant of fibroblast growth factor receptor 2 (Fgfr2) regulates left-right asymmetry in zebrafish

Many organs in vertebrates are left-right asymmetrical located. For example, liver is at the right side and stomach is at the left side in human. Fibroblast growth factor (Fgf) signaling is important for left-right asymmetry. To investigate the roles of Fgfr2 signaling in zebrafish left-right asymmetry, we used splicing blocking morpholinos to specifically block the splicing of fgfr2b and fgfr2c variants, respectively. We found that the relative position of the liver and the pancreas were disrupted in fgfr2c morphants. Furthermore, the left-right asymmetry of the heart became random. Expression pattern of the laterality controlling genes, spaw and pitx2c, also became random in the morphants. Furthermore, lefty1 was not expressed in the posterior notochord, indicating that the molecular midline barrier had been disrupted. It was also not expressed in the brain diencephalon. Kupffer's vesicle (KV) size became smaller in fgfr2c morphants. Furthermore, KV cilia were shorter in fgfr2c morphants. We conclude that the fgfr2c isoform plays an important role in the left-right asymmetry during zebrafish development.     

Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia

Stimulation of astrocytes with the excitatory neurotransmitter glutamate leads to the formation of inositol 1,4,5-trisphosphate and the subsequent increase of intracellular calcium content. Astrocytes express both ionotropic receptors and metabotropic glutamate (mGlu) receptors, of which mGlu5 receptors are probably involved in glutamate-induced calcium signaling. The mGlu5 receptor occurs as two splice variants, mGlu5a and mGlu5b, but it was hitherto unknown which splice variant is responsible for the glutamate-induced effects in astrocytes. We report here that both mRNAs encoding mGlu5 receptor splice variants are expressed by cultured astrocytes. The expression of mGlu5a receptor mRNA is much stronger than that of mGlu5b receptor mRNA in these cells. In situ hybridization experiments reveal neuronal expression of mGlu5b receptor mRNA in adult rat forebrain but a strong neuronal expression of mGlu5a mRNA only in olfactory bulb. Signals for mGlu5a receptor mRNA in the rest of the brain were diffuse and weak but consistently above background. Activation of mGlu5 receptors in astrocytes yields increases in inositol phosphate production and transient calcium responses. It is surprising that the rank order of agonist potency [quisqualate > (2S,1 'S,2'S)-2-(carboxycyclopropyl)glycine = trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) > glutamate] differs from that reported for recombinantly expressed mGlu5a receptors. The expression of mGlu5a receptor mRNA and the occurrence of 1S,3R-ACPD-induced calcium signaling were found also in cultured microglia, indicating for the first time expression of mGlu5a receptors in these macrophage-like cells.