scholarly journals N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation

2014 ◽  
Vol 462 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Brandon E. Aubol ◽  
Ryan M. Plocinik ◽  
Malik M. Keshwani ◽  
Maria L. McGlone ◽  
Jonathan C. Hagopian ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Sr Proteins ◽  
Sr Protein ◽  
N Terminus

The N-terminus of the protein kinase CLK1 induces hyperphosphorylation of SR proteins.

scholarly journals Mobilization of a splicing factor through a nuclear kinase–kinase complex

2018 ◽  
Vol 475 (3) ◽  
pp. 677-690 ◽  
Author(s):  
Brandon E. Aubol ◽  
Malik M. Keshwani ◽  
Laurent Fattet ◽  
Joseph A. Adams
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Sr Proteins ◽  
Splicing Factor ◽  
Nuclear Speckles ◽  
Down Regulation ◽  
Sr Protein ◽  
Protein Activation

The splicing of mRNA is dependent on serine-arginine (SR) proteins that are mobilized from membrane-free, nuclear speckles to the nucleoplasm by the Cdc2-like kinases (CLKs). This movement is critical for SR protein-dependent assembly of the macromolecular spliceosome. Although CLK1 facilitates such trafficking through the phosphorylation of serine-proline dipeptides in the prototype SR protein SRSF1, an unrelated enzyme known as SR protein kinase 1 (SRPK1) performs the same function but does not efficiently modify these dipeptides in SRSF1. We now show that the ability of SRPK1 to mobilize SRSF1 from speckles to the nucleoplasm is dependent on active CLK1. Diffusion from speckles is promoted by the formation of an SRPK1–CLK1 complex that facilitates dissociation of SRSF1 from CLK1 and enhances the phosphorylation of several serine-proline dipeptides in this SR protein. Down-regulation of either kinase blocks EGF-stimulated mobilization of nuclear SRSF1. These findings establish a signaling pathway that connects SRPKs to SR protein activation through the associated CLK family of kinases.

scholarly journals Molecular Mechanism of SR Protein Kinase 1 Inhibition by the Herpes Virus Protein ICP27

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Richard B. Tunnicliffe ◽  
William K. Hu ◽  
Michele Y. Wu ◽  
Colin Levy ◽  
A. Paul Mould ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Alternative Splicing ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Molecular Mechanism ◽  
Sr Proteins ◽  
Sr Protein ◽  
High Affinity ◽  
Simplex Virus

ABSTRACT Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo. Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity. IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

scholarly journals Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

2003 ◽  
Vol 23 (12) ◽  
pp. 4139-4149 ◽  
Author(s):  
Jayendra Prasad ◽  
James L. Manley
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Cell Types ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Sr Protein ◽  
Opposite Pattern ◽  
Founding Member ◽  
Dual Specificity ◽  
Different Cell Types

ABSTRACT SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

Status of Ca2+/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion

1999 ◽  
Vol 277 (3) ◽  
pp. C384-C391 ◽  
Author(s):  
Thomas Netticadan ◽  
Rana Temsah ◽  
Mitsuru Osada ◽  
Naranjan S. Dhalla
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Sr Proteins ◽  
Dependent Protein Kinase ◽  
Sr Protein ◽  
Phosphatase Activities ◽  
The Status ◽  
Dependent Protein

Although the sarcoplasmic reticulum (SR) is known to regulate the intracellular concentration of Ca2+ and the SR function has been shown to become abnormal during ischemia-reperfusion in the heart, the mechanisms for this defect are not fully understood. Because phosphorylation of SR proteins plays a crucial role in the regulation of SR function, we investigated the status of endogenous Ca2+/calmodulin-dependent protein kinase (CaMK) and exogenous cAMP-dependent protein kinase (PKA) phosphorylation of the SR proteins in control, ischemic (I), and ischemia-reperfused (I/R) hearts treated or not treated with superoxide dismutase (SOD) plus catalase (CAT). SR and cytosolic fractions were isolated from control, I, and I/R hearts treated or not treated with SOD plus CAT, and the SR protein phosphorylation by CaMK and PKA, the CaMK- and PKA-stimulated Ca2+ uptake, and the CaMK, PKA, and phosphatase activities were studied. The SR CaMK and CaMK-stimulated Ca2+ uptake activities, as well as CaMK phosphorylation of Ca2+ pump ATPase (SERCA2a) and phospholamban (PLB), were significantly decreased in both I and I/R hearts. The PKA phosphorylation of PLB and PKA-stimulated Ca2+ uptake were reduced significantly in the I/R hearts only. Cytosolic CaMK and PKA activities were unaltered, whereas SR phosphatase activity in the I and I/R hearts was depressed. SOD plus CAT treatment prevented the observed alterations in SR CaMK and phosphatase activities, CaMK and PKA phosphorylations, and CaMK- and PKA-stimulated Ca2+ uptake. These results indicate that depressed CaMK phosphorylation and CaMK-stimulated Ca2+ uptake in I/R hearts may be due to a depression in the SR CaMK activity. Furthermore, prevention of the I/R-induced alterations in SR protein phosphorylation by SOD plus CAT treatment is consistent with the role of oxidative stress during ischemia-reperfusion injury in the heart.

scholarly journals Functional properties of p54, a novel SR protein active in constitutive and alternative splicing.

1996 ◽  
Vol 16 (10) ◽  
pp. 5400-5408 ◽  
Author(s):  
W J Zhang ◽  
J Y Wu
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Sr Protein ◽  
Protein Protein Interaction ◽  
Small Nuclear Ribonucleoprotein ◽  
S100 Extract ◽  
U2 Auxiliary Factor ◽  
Auxiliary Factor

The p54 protein was previously identified by its reactivity with an autoantiserum. We report here that p54 is a new member of the SR family of splicing factors, as judged from its structural, antigenic, and functional characteristics. Consistent with its identification as an SR protein, p54 can function as a constitutive splicing factor in complementing splicing-deficient HeLa cell S100 extract. However, p54 also shows properties distinct from those of other SR family members, p54 can directly interact with the 65-kDa subunit of U2 auxiliary factor (U2AF65), a protein associated with the 3' splice site. In addition, p54 interacts with other SR proteins but does not interact with the U1 small nuclear ribonucleoprotein U1-70K or the 35-kDa subunit of U2 auxiliary factor (U2AF35). This protein-protein interaction profile is different from those of prototypical SR proteins SC35 and ASF/SF2, both of which interact with U1-70K and U2AF35 but not with U2AF65. p54 promotes the use of the distal 5' splice site in E1A pre-mRNA alternative splicing, while the same site is suppressed by ASF/SF2 and SC35. These findings and the differential tissue distribution of p54 suggest that this novel SR protein may participate in regulation of alternative splicing in a tissue- and substrate-dependent manner.

Tyrosine-protein kinase Src regulates Kv1.5 channel activity and membrane expression through interaction with the N-terminus of the channel

2020 ◽  
Author(s):  
Taylore Dodd ◽  
Tingzhong Wang ◽  
Shetuan Zhang
Keyword(s):  
Protein Kinase ◽  
Cell Voltage ◽  
Underlying Mechanism ◽  
Delayed Rectifier ◽  
Binding Motif ◽  
Membrane Expression ◽  
Binding Motifs ◽  
Delayed Rectifier Potassium Current ◽  
N Terminus ◽  
Tyrosine Protein Kinase

Kv1.5 is a voltage-gated potassium channel that generates the ultra-rapid delayed rectifier potassium current (IKur) important in the repolarization of the atrial action potential. Malfunction of the Kv1.5 channel often results in atrial fibrillation (AFib). A reduction in Kv1.5 current (IKv1.5) occurs upon activation of the endogenous tyrosine-protein kinase Src. The Src SH3 domain binds to proline-rich motifs located within the N-terminus of Kv1.5. Disruption of these binding motifs has been involved in the development of familial AFib. The mechanism underlying the reduction of IKv1.5 upon Src activation has not yet been established and the relationship between Kv1.5 and Src is poorly understood. Therefore, the present study aims to further elucidate the mechanism behind IKv1.5  reduction. The hypothesis that Src regulates Kv1.5 activity by altering the density of mature membrane-localized channels was tested using whole-cell voltage clamp and Western blot analysis. We demonstrate that Src tonically inhibits Kv1.5 activity and decreases the density of mature membrane-localized channels. Kv1.5 channels possessing mutations within the Src binding motifs were also investigated and it was determined that each binding motif contributes to the Kv1.5-Src relationship, however, the binding of Src to an individual motif is sufficiently effective. Our findings indicate that Src regulates Kv1.5 through an interaction with the N-terminal binding motifs and suggests that the inhibition of forward trafficking may be involved in the underlying mechanism. (Supported by the Heart and Stroke foundation of Canada and The Canadian Institutes of Health Research).

scholarly journals Screening for Modulators of Spermine Tolerance Identifies Sky1, the SR Protein Kinase of Saccharomyces cerevisiae, as a Regulator of Polyamine Transport and Ion Homeostasis

2001 ◽  
Vol 21 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Omri Erez ◽  
Chaim Kahana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Osmotic Shock ◽  
Ion Homeostasis ◽  
Inorganic Ions ◽  
Yeast Cells ◽  
Wild Type ◽  
Sr Protein ◽  
Polyamine Transport ◽  
Increased Sensitivity

ABSTRACT Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants ofSaccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Δ cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity ofPPZ1 but not of ENA1.

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