The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.

Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.     

Antagonistic effects of the SRp30c protein and cryptic 5' splice sites on the alternative splicing of the apoptotic regulator Bcl-x

Alternative 5' splice site selection allows Bcl-x to produce two isoforms with opposite effects on apoptosis. The pro-apoptotic Bcl-x(S) variant is up-regulated by ceramide and down-regulated by protein kinase C through specific cis-acting exonic elements, one of which is bound by SAP155. Splicing to the Bcl-x(S) 5' splice site is also enforced by heterogeneous nuclear ribonucleoprotein (hnRNP) F/H proteins and by Sam68 in cooperation with hnRNP A1. Here, we have characterized exon elements that influence splicing to the 5' splice site of the anti-apoptotic Bcl-x(L) isoform. Within a 86-nucleotide region (B3) located immediately upstream of the Bcl-x(L) donor site we have identified two elements (ML2 and AM2) that stimulate splicing to the Bcl-x(L) 5' splice site. SRp30c binds to these elements and can shift splicing to the 5' splice site of Bcl-x(L) in an ML2/AM2-dependent manner in vitro and in vivo. The B3 region also contains an element that represses the use of Bcl-x(L). This element is bound by U1 small nuclear ribonucleoprotein and contains two 5' splice sites that can be used when the Bcl-x(L) 5' splice site is mutated or the ML2/AM2 elements are deleted. Conversely, mutating the cryptic 5' splice sites stimulates splicing to the Bcl-x(L) site. Thus, SRp30c stimulates splicing to the downstream 5' splice site of Bcl-x(L), thereby attenuating the repressive effect of upstream U1 snRNP binding sites.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

Antibodies elicited by N2,N2-7-trimethylguanosine react with small nuclear RNAs and ribonucleoproteins

The 5' ends of U1, U2, U3, U4, and U5 small nuclear RNAs (snRNA) are capped by a structure which contains N2,N2-7-trimethylguanosine (m2,2,7 G). m2,2,7 G was used as hapten to raise antibodies in rabbits, and these antibodies were linked to Sepharose. When deproteinized RNA was passed through this antibody column, these snRNA species were retained by the column. Conversely, 4 S, 5 S, 5.8 S, U6, and 7 S RNA, whose 5' termini do not contain m2,2,7 G, were not recognized. After a nuclear extract was loaded on the column, U1 RNA and some U2 RNA were retained. Therefore, the 5' ends of at least U1 RNA are accessible when this RNA species is in small nuclear ribonucleoprotein particle (snRNP) form. This is of interest, since it has been proposed that the 5' terminus sequence of U1 RNA may hybridize with splice junctions in heterogeneous nuclear ribonucleoprotein particles (hnRNP) during mRNA splicing. The retention of m2,2,7 G-containing RNA species by these antibodies is not due to association of snRNAs or snRNPs with heterogeneous nuclear RNA (hnRNA) or hnRNP (and antibody recognition of 7-monomethylguanosine residues in hnRNA), since the reaction still occurs after removal of hnRNA or hnRNP by sucrose gradient centrifugation.     

Membrane depolarization induces the undulating phosphorylation/dephosphorylation of glycogen synthase kinase 3beta, and this dephosphorylation involves protein phosphatases 2A and 2B in SH-SY5Y human neuroblastoma cells

Changes in plasma membrane electrical potential evoke signals that regulate the expressions of various genes in the nervous system. However, the role of glycogen synthase kinase 3beta (GSK-3beta) in this process has not been elucidated. Thus, this study was performed to examine whether membrane depolarization can regulate the phosphorylation of GSK-3beta and to identify the molecular mechanisms involved in this regulation. The depolarization by treating with 100 mm KCl for 5 min resulted in the undulating phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells, in H19 -7/IGF-IR rat embryonic hippocampal cells, and in PC12 rat pheochromocytoma cells, but not in A172 human glioblastoma cells. Cellular beta-catenin contents showed a temporal pattern similar to that of the Ser-9 phosphorylation of GSK-3beta. Treatment with wortmannin or calphostin C or the expression of dominant negative Akt inhibited phosphorylation of GSK-3beta at Ser-9 following the KCl-induced depolarization of SH-SY5Y cells. Moreover, pretreatment with okadaic acid or cyclosporin A blocked the dephosphorylation of GSK-3beta at Ser-9 at 0, 15, and 30 min after KCl-induced depolarization, and the activity of protein phosphatases (PP) 2A and 2B increased at these times. Treatment with nifedipine or calcium-free medium inhibited GSK-3beta dephosphorylation following membrane depolarization, and the amounts of co-immunoprecipitated GSK-3beta and PP2A changed in parallel with GSK-3beta dephosphorylation. Our study demonstrated that KCl-induced depolarization caused undulating GSK-3beta phosphorylation/dephosphorylation, which was regulated for the most part by phosphatidylinositol 3-kinase and Akt (phosphorylation) and PP2A and PP2B (dephosphorylation), respectively.     

Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the beta(2) subunit

Activation of protein kinase A (PKA) through the beta-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the beta(2) subunit are of functional significance, we coexpressed wild-type (WT) or mutant beta(2) subunits in tsA-201 cells together with an alpha(1C) subunit, alpha(1C)Delta1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated alpha(1C) subunit was similar to the truncated alpha(1C) subunit isolated from cardiac tissue not only in size ( approximately 190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT beta(2) subunit, voltage-activated Ba(2+) currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the beta(2) subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca(2+) currents may be at least partially caused by phosphorylation of the beta(2) subunit at Ser-478 and Ser-479.     

Ascofuranone prevents ER stress-induced insulin resistance via activation of AMP-activated protein kinase in L6 myotube cells

The current study presents that ascofuranone isolated from a phytopathogenic fungus, Ascochyta viciae, has antitumor activity against various transplantable tumors and a considerable hypolipidemic activity. AMP-activated protein kinase (AMPK) plays a critical role in cellular glucose and lipid homeostasis. We found that ascofuranone improves ER stress-induced insulin resistance by activating AMPK through the LKB1 pathway. In L6 myotube cells, ascofuranone treatment increased the phosphorylation of the Thr-172 residue of the AMPKα subunit and the Ser-79 subunit of acetyl-CoA carboxylase (ACC) and cellular glucose uptake. Ascofuranone-induced phosphorylation of AMPK and ACC was not increased in A549 cells lacking LKB1. Interestingly, ascofuranone treatment also improved insulin signaling impaired by ER stress in L6 myotube cells. These effects were all reversed by pretreatment with Compound C, an AMPK inhibitor or with adenoviral-mediated dominant-negative AMPKα2. Taken together, these results indicated that ascofuranone-mediated enhancement of glucose uptake and reduction of impaired insulin sensitivity in L6 cells is predominantly accomplished by activating AMPK, thereby mediating beneficial effects in type 2 diabetes and insulin resistance.     

RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells

17beta-Estradiol (E(2)) reduces oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3). The mechanism by which the nongenomic effects of E(2) contributed to the protection against mitochondrial membrane depolarization was investigated. Mitochondrial membrane integrity is regulated by phosphorylation of BAD, and it is known that phosphorylation of Ser(112) inactivates BAD and prevents its participation in the mitochondrial death pathway. We found that E(2) rapidly increased both the phosphorylation of ERK2 and Ser(112) in BAD. Ser(112) is phosphorylated by p90 ribosomal S6 kinase (RSK), a Ser/Thr kinase, which is a downstream effector of ERK1/2. Inhibition of RSK by the RSK-specific inhibitor SL0101 did not reduce the level of E(2)-induced phosphorylation of Ser(112). Silencing BAD using small interfering RNA did not alter mitochondrial membrane depolarization elicited by peroxide insult. However, under the same conditions, silencing ERK2 dramatically increased membrane depolarization compared with the control small interfering RNA. Therefore, ERK2, functioning through a BAD-independent mechanism regulates MMP in humans lens epithelial cells. We propose that estrogen-induced activation of ERK2 acts to protect cells from acute oxidative stress. Moreover, despite the fact that ERK2 plays a regulatory role in mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism.     

Signalling in taste receptor cells: cAMP-dependent protein kinase causes depolarization by closure of 44 pS K-channels

1. In whole-cell patch-clamp recordings cytosolic cAMP causes a substantial depolarization of taste receptor cells isolated from the frog tongue. The depolarization requires the presence of ATP in the cell and is suppressed by protein kinase inhibitor. 2. The depolarization also develops in the absence of cAMP while the catalytic subunit of the kinase is allowed to diffuse into the cell in the presence of ATP. 3. In membrane patches excised from these cells the catalytic subunit is found to inactivate K-channels of 44 pS conductance, presumably by phosphorylation. 4. It appears that cAMP is one of the intracellular messengers in gustatory chemoreception, and that it causes membrane depolarization through activation of a protein kinase which controls the activity of one set of K-channels.     

Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons

Splicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function. Thus hnRNP M, along with other members of the hnRNP family of RNA-binding proteins, plays a combinatorial role in regulation of FGFR2 alternative splicing. We also determined that hnRNP M can affect the splicing of several other alternatively spliced exons. This activity of hnRNP M included the ability not only to induce exon skipping but also to promote exon inclusion. This is the first report demonstrating a role for this abundant hnRNP family member in alternative splicing in mammals and suggests that this protein may broadly contribute to the fidelity of splice site recognition and alternative splicing regulation.     

Multiple phosphorylations of cytochrome c oxidase and their functions

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial electron transport chain, is regulated by isozyme expression, allosteric effectors such as the ATP/ADP ratio, and reversible phosphorylation. Of particular interest is the "allosteric ATP-inhibition," which has been hypothesized to keep the mitochondrial membrane potential at low healthy values (<140 mV), thus preventing the formation of superoxide radical anions, which have been implicated in multiple degenerative diseases. It has been proposed that the "allosteric ATP-inhibition" is switched on by the protein kinase A-dependent phosphorylation of COX. The goal of this study was to identify the phosphorylation site(s) involved in the "allosteric ATP-inhibition" of COX. We report the mass spectrometric identification of four new phosphorylation sites in bovine heart COX. The identified phosphorylation sites include Tyr-218 in subunit II, Ser-1 in subunit Va, Ser-2 in subunit Vb, and Ser-1 in subunit VIIc. With the exception of Ser-2 in subunit Vb, the identified phosphorylation sites were found in enzyme samples with and without "allosteric ATP inhibition," making Ser-2 of subunit Vb a candidate site enabling allosteric regulation. We therefore hypothesize that additional phosphorylation(s) may be required for the "allosteric ATP-inhibition," and that these sites may be easily dephosphorylated or difficult to identify by mass spectrometry.     

Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine–serine-rich (RS) domains of SR- and SR-like proteins

The Fe(II) and 2-oxoglutarate dependent oxygenase Jmjd6 has been shown to hydroxylate lysine residues in the essential splice factor U2 auxiliary factor 65 kDa subunit (U2AF65) and to act as a modulator of alternative splicing. We describe further evidence for the role of Jmjd6 in the regulation of pre-mRNA processing including interactions of Jmjd6 with multiple arginine–serine-rich (RS)-domains of SR- and SR-related proteins including U2AF65, Luc7-like protein 3 (Luc7L3), SRSF11 and Acinus S′, but not with the bona fide RS-domain of SRSF1. The identified Jmjd6 target proteins are involved in different mRNA processing steps and play roles in exon dependent alternative splicing and exon definition. Moreover, we show that Jmjd6 modifies splicing of a constitutive splice reporter, binds RNA derived from the reporter plasmid and punctually co-localises with nascent RNA. We propose that Jmjd6 exerts its splice modulatory function by interacting with specific SR-related proteins during splicing in a RNA dependent manner.     

MYPT1 protein isoforms are differentially phosphorylated by protein kinase G

Smooth muscle relaxation in response to NO signaling is due, in part, to a Ca(2+)-independent activation of myosin light chain (MLC) phosphatase by protein kinase G Iα (PKGIα). MLC phosphatase is a trimeric complex of a 20-kDa subunit, a 38-kDa catalytic subunit, and a 110-133-kDa myosin-targeting subunit (MYPT1). Alternative mRNA splicing produces four MYPT1 isoforms, differing by the presence or absence of a central insert and leucine zipper (LZ). The LZ domain of MYPT1 has been shown to be important for PKGIα-mediated activation of MLC phosphatase activity, and changes in LZ+ MYPT1 isoform expression result in changes in the sensitivity of smooth muscle to NO-mediated relaxation. Furthermore, PKGIα has been demonstrated to phosphorylate Ser-694 of MYPT1, but phosphorylation at this site does not always accompany cGMP-mediated smooth muscle relaxation. This study was designed to determine whether MYPT1 isoforms are differentially phosphorylated by PKGIα. The results demonstrate that purified LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694, whereas fragments lacking the LZ domain are poor PKGIα substrates. Mutation of Ser-667 and Ser-694 to Ala and/or Asp showed that Ser-667 phosphorylation is more rapid than Ser-694 phosphorylation, suggesting that Ser-667 may play an important role in the activation of MLC phosphatase. These results demonstrate that MYPT1 isoform expression is important for determining the heterogeneous response of vascular beds to NO and NO-based vasodilators, thereby playing a central role in the regulation of vascular tone in health and disease.     

Regulatory roles of heterogeneous nuclear ribonucleoprotein M and Nova-1 protein in alternative splicing of dopamine D2 receptor pre-mRNA

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.