Mass Spectrometric and Kinetic Analysis of ASF/SF2 Phosphorylation by SRPK1 and Clk/Sty

Velazquez-Dones, Adolfo; Hagopian, Jonathan C.; Ma, Chen‐Ting; Zhong, Xiang-Yang; Zhou, Huilin; Ghosh, Gourisankar; Fu, Xiang‐Dong; Adams, Joseph A.

doi:10.1074/jbc.m504156200

Cited by 90 publications

(141 citation statements)

References 32 publications

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“…The SR protein family can be further extended by inclusion of a structurally and functionally diverse group of nuclear proteins that possess RS repeats but lack RRM domains (11). The RS domains are processively phosphorylated on their Ser residues by a set of dedicated kinases (12)(13)(14)(15)(16). Phosphorylation of SR proteins is thought to trigger their import into the nucleus and is subsequently required for spliceosome assembly, whereas their dephosphorylation allows for splicing to proceed (17)(18)(19)(20).…”

mentioning

confidence: 99%

Structural basis for nuclear import of splicing factors by human Transportin 3

Maertens

Cook

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

167

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Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginineserine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran-and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.T he transport of macromolecules between cytoplasm and nucleus is orchestrated by a family of nuclear import and export receptors (1). Referred to as importins and exportins, these proteins bind their specific cargoes and translocate them across the nuclear pore complex. The process is regulated by the small GTPase Ran that partitions between cytoplasm and nucleus in the predominantly GDP-and GTP-bound form, respectively. Importins associate with their cargoes in the cytoplasm, and the competitive binding of RanGTP induces them to release their cargoes in the nucleus (2). Most nuclear import/export receptors belong to the β-karyopherin family of proteins, with 22 members encoded in the human genome (3). The majority of β-karyopherins bind their cargoes directly, recognizing a linear nuclear localization or export signal and/or a specific tertiary/quaternary structural feature (4, 5).A fundamentally important type of a nuclear localization signal (NLS), comprising sequences rich in Arg-Ser and/or ArgAsp/Glu/Gly dipeptides (referred to as RS or RS-like domains), belongs to the family of Ser/Arg-rich (SR) proteins. These nuclear proteins also contain RNA recognition motif (RRM) domains and play essential roles in pre-mRNA splicing and 3′ processing and participate in transcription regulation, mRNA transport, translation, and nonsense-mediated mRNA decay (6). The splicing factors alternative splicing factor/splicing factor 2 (ASF/SF2) and SC35 along with the cleavage and polyadenylation specificity factor 6 (CPSF6, also known as CF-Im-68) are among the best-characterized metazoan SR proteins (7-10). The SR protein family can be further extended by inclusion of a structurally and functionally diverse group of nuclear proteins that possess RS repeats but lack RRM domains (11). The RS domains are processively phosphorylated on their Ser residues by a set of dedicated kinases (12-16). Phosphorylation of SR proteins is thought to...

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mentioning

confidence: 99%

Structural basis for nuclear import of splicing factors by human Transportin 3

Maertens

Cook

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

167

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“…Mass spectrometric analysis showed that SRPK1 phosphorylates approximately 10 sites in the N-terminal half of the RS domain (RS1), while Clk/Sty phosphorylates the RS1 sites as well as the remaining serines in the C-terminal portion of the RS domain (RS2) [71]. Start-trap experiments showed that both SRPK1 and Clk/Sty kinases carry out phosphorylation of the RS domain in a processive manner.…”

Section: Processive Phosphorylation Of Asf/sf2mentioning

confidence: 99%

“…Recent reports have shown that ASF/SF2 is phosphorylated by SRPK1 and Clk/Sty kinases in a fully processive manner [71,72]. SRPK1 binding to ASF/SF2 is dependent on the phosphorylation state of ASF/SF2 [73].…”

Section: Processive Phosphorylation Of Asf/sf2mentioning

confidence: 99%

Processive phosphorylation: Mechanism and biological importance

Patwardhan

Miller

2007

Cellular Signalling

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Recent proteomic data indicate that a majority of the phosphorylated proteins in a eucaryotic cell contain multiple sites of phosphorylation. In many signaling events, a single kinase phosphorylates multiple sites on a target protein. Processive phosphorylation occurs when a protein kinase binds once to a substrate and phosphorylates all of the available sites before dissociating. In this review, we discuss examples of processive phosphorylation by serine/threonine kinases and tyrosine kinases. We describe current experimental approaches for distinguishing processive from non-processive phosphorylation. Finally, we contrast the biological situations that are suited to regulation by processive and non-processive phosphorylation.

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“…The cloning of ASF/SF2 in Escherichia coli expression vector has been described earlier (11). The expression vector was transformed into E. coli BL21(DE3) cells (Invitrogen) and grown in LB containing 150 g/ml ampicillin.…”

Section: Cloning and Expression Of Recombinant Proteins-expressionmentioning

confidence: 99%

“…1a). SRPK1 phosphorylates ϳ10 -12 of these serines in the N-terminal RS1 region of the RS domain (11). This multisite phosphorylation occurs in a processive manner where the kinase remains bound to the substrate until all sites are phosphorylated (12).…”

mentioning

confidence: 99%

Structurally Unique Yeast and Mammalian Serine-Arginine Protein Kinases Catalyze Evolutionarily Conserved Phosphorylation Reactions

Lukasiewicz

Velazquez-Dones

Huynh

et al. 2007

Journal of Biological Chemistry

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The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and ϳ12 of these serines are processively phosphorylated by the SR protein kinase 1 (SRPK1). We have recently shown that a docking motif in ASF/SF2 specifically interacts with a groove in SRPK1, and this interaction is necessary for processive phosphorylation. We previously showed that SRPK1 and its yeast ortholog Sky1p maintain their active conformations using diverse structural strategies. Here we tested if the mechanism of ASF/SF2 phosphorylation by SRPK is evolutionarily conserved. We show that Sky1p forms a stable complex with its heterologous mammalian substrate ASF/SF2 and processively phosphorylates the same sites as SRPK1. We further show that Sky1p utilizes the same docking groove to bind yeast SR-like protein Gbp2p and phosphorylates all three serines present in a contiguous RS dipeptide stretch. However, the mechanism of Gbp2p phosphorylation appears to be non-processive. Thus, there are physical attributes of SR and SR-like substrates that dictate the mechanism of phosphorylation, whereas the ability to processively phosphorylate substrates is inherent to SR protein kinases.Pre-mRNA splicing and mRNA export are complex processes, which involve a large number of protein and RNA factors (1-4). SR proteins, a class of non-small nuclear ribonucleoprotein splicing factors, participate in every step in the spliceosome assembly and catalysis (5). SR proteins have unique domain architecture. The N-terminal domain contains one or two RNA recognition motifs (RRMs) 3 and is responsible for RNA binding, and the C-terminal domain, known as the RS domain, is rich in long stretches of serine-arginine/arginineserine (SR/RS) dipeptides (6). The RS domains of SR proteins are extensively phosphorylated. Although many kinases have been demonstrated to target SR proteins, members of the SR protein kinase (SRPK) family have been established as the predominant kinase (6 -10). Mammalian SR protein kinase 1 (SRPK1) and the yeast enzyme, Sky1p, are the two most studied SRPKs. Members of the SRPK family display strict substrate specificity, preferring to phosphorylate only serine residues flanked by arginines. One of the well studied mammalian SR proteins, ASF/SF2, contains 20 serines within its 50-residuelong RS domain (see Fig. 1a). SRPK1 phosphorylates ϳ10 -12 of these serines in the N-terminal RS1 region of the RS domain (11). This multisite phosphorylation occurs in a processive manner where the kinase remains bound to the substrate until all sites are phosphorylated (12). Phosphorylation of RS1 is essential for the nuclear import of ASF/SF2.In yeast, Sky1p regulates the phosphorylation and nuclear import of SR-like proteins . However, unlike the mammalian SR proteins, the yeast proteins do not have a classic RS domain with long stretches of arginine/ serine repeats (6, 16) (Fig. 1a). In the case of Npl3p, eight isolated RS dipeptides are present within the RGG domain, and the C-terminal most RS dipeptide is...

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Mass Spectrometric and Kinetic Analysis of ASF/SF2 Phosphorylation by SRPK1 and Clk/Sty

Cited by 90 publications

References 32 publications

Structural basis for nuclear import of splicing factors by human Transportin 3

Structural basis for nuclear import of splicing factors by human Transportin 3

Processive phosphorylation: Mechanism and biological importance

Structurally Unique Yeast and Mammalian Serine-Arginine Protein Kinases Catalyze Evolutionarily Conserved Phosphorylation Reactions

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