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

Prasad, J.K.; Manley, James L.

doi:10.1128/mcb.23.12.4139-4149.2003

Cited by 65 publications

(55 citation statements)

References 61 publications

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“…The SRPK1 reaction is accompanied by a time-dependent increase in band intensity for ASF/SF2 as previously described (21). The Clk/Sty reaction is accompanied by both an increase in band intensity and a small change in migration state of ASF/ SF2 on the gel as described previously (26). Data in Fig.…”

Section: Resultssupporting

confidence: 59%

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

Velazquez-Dones

Hagopian

et al. 2005

Journal of Biological Chemistry

136

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Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.It is estimated that more than one-half of human genes are alternatively spliced (1). Although many of these genes may contain only a few splice variants, a few have been shown to possess thousands (1-3). RNA splicing occurs in the nucleus at a macromolecular complex composed of many RNA and protein molecules known as the spliceosome (4, 5). In the last decade it has become apparent that both the assembly of the spliceosome and selection of splice sites depend on the proper phosphorylation of a family of splicing factors known as SR proteins (1). The SR proteins are so named because they have long stretches of arginineserine dipeptide repeats in RS domains. Phosphorylation leads to translocation of the SR protein from the cytoplasm to the nucleus (6, 7) and recruitment of the SR proteins from sites of nuclear storage in speckles to nascent transcripts for splicing (8 -10). Phosphorylated SR proteins are believed to facilitate both 5Ј and 3Ј splice site recognition through interaction with the RS domain in U1-70 K (a component of the U1 small nuclear ribonucleoprotein) and the U2AF heterodimer (11-13). More recently, RS domains were shown to interact directly with critical cis-acting elements in pre-mRNA (14,15). In addition to their functions in the spliceosome, SR proteins are also shown to play a role in the export of processed mRNA from the nucleus to the cytoplasm for translation. Two shuttling SR proteins, ASF/SF2 6 and 9G8, have been shown to conduct the export of mRNA through interactions with a nuclear export factor, TAP (16 -18). Hypophosphorylation of these SR proteins promotes interaction with TAP suggesting that transport may require splicing factor dephosphorylation. These shuttling SR proteins were also found to play a direct role in translation in the cytoplasm (19).Although it is clear that SR proteins can be polyphosphorylated in the RS domain regions by the SRPK ...

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Section: Resultssupporting

confidence: 59%

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

Velazquez-Dones

Hagopian

et al. 2005

Journal of Biological Chemistry

136

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“…The importance of this translocation is not known, although it likely permits the kinase access to its substrates, including the SR proteins contained within nuclear speckles (30), and the LBR (36). Other regulatory mechanisms of SRPK1 activity could include autophosphorylation of the kinase, as is apparent for CLK family kinases, which also phosphorylate the RS domains of SR factors, and is found to modulate substrate recognition and lead to changes in the phosphorylation patterns on specific SR proteins (41). It is therefore FIG.…”

Section: Discussionmentioning

confidence: 99%

The E1^E4 Protein of Human Papillomavirus Interacts with the Serine-Arginine-Specific Protein Kinase SRPK1

Bell

Martin

Roberts

2007

J Virol

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Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1 E4 protein during the productive phase of infection. However, the precise mechanisms of how E1 E4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1 E4. We map critical residues within an arginine-rich domain of HPV1 E1 E4, and in a region known to facilitate E1 E4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1 E4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1 E4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1 E4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1 E4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1 E4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.Human papillomaviruses (HPVs) comprise a family of small double-stranded DNA viruses that have a tropism for human epithelial cells at skin and mucosal surfaces. To date, based on genome sequence, over 100 HPV types have been described, and these virus types vary in both their anatomical tropism and their potential to induce malignant transformation (10). The HPV replication cycle is intimately linked with the differentiation of the host epithelium (21). To initiate an infection, the virus requires access, typically via a cut or abrasion, to basal cells where the virus maintains itself at a low copy number. Subsequently, infected cells undergo differentiation and migrate to the epithelial surface, which initiates the productive phase of virus infection, signified by genome amplification, the synthesis of structural proteins, and assembly of infectious progeny. These events are orchestrated by both transcriptional and posttranscriptional mechanisms and consequently ensure that structural proteins are only synthesized in terminally differentiated cells (61).High-level expression of the E4 protein accompanies the productive phase of the HPV life cycle (40). E4 is translated from spliced E1 E4 transcripts and encodes the first 5 amino acids from the N terminus of the E1 protein fused to the E4 coding sequence. The E4 open reading frame (ORF) is the most divergent ORF within the HPV family. While there is sequence homology between E1 E4 proteins, this is generally restricted to virus types ...

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“…Results in cultured mammalian cells initially described LAMMER kinases as exclusively nuclear (Colwill et al 1996b;Nayler et al 1997Nayler et al , 1998, but subsequent studies have shown that the mammalian CLK kinases are also cytoplasmic (Moeslein et al 1999;Menegay et al 2000;Prasad and Manley 2003), suggesting the possibility of shuttling between the two intracellular compartments. At least six Doa isoforms are produced via alternative promoter utilization (Kpebe and Rabinow 2008a), of Supporting information is available online at http:/ /www.genetics.org/ cgi/content/full/genetics.109.109553/DC1.…”

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confidence: 99%

Drosophila Translational Elongation Factor-1γ Is Modified in Response to DOA Kinase Activity and Is Essential for Cellular Viability

et al. 2010

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Drosophila translational elongation factor-1g (EF1g) interacts in the yeast two-hybrid system with DOA, the LAMMER protein kinase of Drosophila. Analysis of mutant EF1g alleles reveals that the locus encodes a structurally conserved protein essential for both organismal and cellular survival. Although no genetic interactions were detected in combinations with mutations in EF1a, an EF1g allele enhanced mutant phenotypes of Doa alleles. A predicted LAMMER kinase phosphorylation site conserved near the C terminus of all EF1g orthologs is a phosphorylation site in vitro for both Drosophila DOA and tobacco PK12 LAMMER kinases. EF1g protein derived from Doa mutant flies migrates with altered mobility on SDS gels, consistent with it being an in vivo substrate of DOA kinase. However, the aberrant mobility appears to be due to a secondary protein modification, since the mobility of EF1g protein obtained from wild-type Drosophila is unaltered following treatment with several nonspecific phosphatases. Expression of a construct expressing a serine-to-alanine substitution in the LAMMER kinase phosphorylation site into the fly germline rescued null EF1g alleles but at reduced efficiency compared to a wild-type construct. Our data suggest that EF1g functions in vital cellular processes in addition to translational elongation and is a LAMMER kinase substrate in vivo.

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

Cited by 65 publications

References 61 publications

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

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

The E1^E4 Protein of Human Papillomavirus Interacts with the Serine-Arginine-Specific Protein Kinase SRPK1

Drosophila Translational Elongation Factor-1γ Is Modified in Response to DOA Kinase Activity and Is Essential for Cellular Viability

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