Novel destabilization of nucleotide binding by the gamma phosphate of ATP in the yeast SR protein kinase Sky1p

SR protein kinases (SRPKs) regulate the temporal and cell-specific selection of alternative splice sites. These enzymes are highly unique members of the protein kinase family. SRPKs contain a large domain insert (approximately 200 residues) within the kinase core, do not require phosphorylation for regulation, have an extended helix insert near the nucleotide pocket, and possess unusual substrate specificity determinants. The yeast SRPK, Sky1p, rapidly phosphorylates its natural substrate Npl3 but binds ATP with a high K(m), suggesting that some of these distinctive structural features may be correlated with nucleotide binding [Aubol et al. (2002) Biochemistry 41, 10002-10009]. To address this issue, the nucleotide binding properties of Sky1p were studied using fluorescence spectroscopy. The affinities of several nucleotides (ATP, ADP, AMP, adenosine, and AMPPNP) to Sky1p and the prototype kinase, cAMP-dependent protein kinase, were compared in the absence and presence of the metal activator, Mg(2+), using a fluorescence-based displacement assay. The data indicate that Sky1p, unlike cAMP-dependent protein kinase, potently destabilizes the gamma phosphate of ATP. This novel finding suggests that rapid phosphoryl transfer may be facilitated by unique mechanisms in both protein kinases.     

Structurally unique yeast and mammalian serine-arginine protein kinases catalyze evolutionarily conserved phosphorylation reactions

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and approximately 12 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.     

Mechanistic insights into Sky1p,a yeast homologue of the mammalian SR protein kinases

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.     

Serine-arginine protein kinases: a small protein kinase family with a large cellular presence

Serine-arginine protein kinases (SPRKs) constitute a relatively novel subfamily of serine-threonine kinases that specifically phosphorylate serine residues residing in serine-arginine/arginine-serine dipeptide motifs. Fifteen years of research subsequent to the purification and cloning of human SRPK1 as a SR splicing factor-phosphorylating protein have lead to the accumulation of information on the function and regulation of the different members of this family, as well as on the genomic organization of SRPK genes in several organisms. Originally considered to be devoted to constitutive and alternative mRNA splicing, SRPKs are now known to expand their influence to additional steps of mRNA maturation, as well as to other cellular activities, such as chromatin reorganization in somatic and sperm cells, cell cycle and p53 regulation, and metabolic signalling. Similarly, SRPKs were considered to be constitutively active kinases, although several modes of regulation of their function have been demonstrated, implying an elaborate cellular control of their activity. Finally, SRPK gene sequence information from bioinformatics data reveals that SRPK gene homologs exist either in single or multiple copies in every single eukaryotic organism tested, emphasizing the importance of SRPK protein function for cellular life.     

The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs)

Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine-serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co-purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.     

Regulated cellular partitioning of SR protein-specific kinases in mammalian cells

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.     

SR protein kinase 1 is resilient to inactivation

SR protein kinase 1 (SRPK1) is a constitutively active kinase, which processively phosphorylates multiple serines within its substrates, ASF/SF2. We describe crystallographic, molecular dynamics, and biochemical results that shed light on how SRPK1 preserves its constitutive active conformation. Our structure reveals that unlike other known active kinase structures, the activation loop remains in an active state without any specific intraprotein interactions. Moreover, SRPK1 remains active despite extensive mutation to the activation segment. Molecular dynamics simulations reveal that SRPK1 partially absorbs the effect of mutations by forming compensatory interactions that maintain a catalytically competent chemical environment. Furthermore, SRPK1 is similarly resistant to deletion of its spacer loop region. Based upon a model of SRPK1 bound to a segment encompassing the docking motif and active-site peptide of ASF/SF2, we suggest a mechanism for processive phosphorylation and propose that the atypical resiliency we observed is critical for SRPK1's processive activity.     

Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase

Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1(+): Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.     

The RGG domain of Npl3p recruits Sky1p through docking interactions

The SR protein kinase in yeast, Sky1p, phosphorylates yeast SR-like protein, Npl3p, at a single serine residue located at its C terminus. We report here the X-ray crystal structure of Sky1p bound to a substrate peptide and ADP. Surprisingly, an Npl3p-derived substrate peptide occupies a groove 20 A away from the kinase active site. In vitro studies support the substrate-docking role of this groove. Mutagenesis and binding studies reveal that multiple degenerate short peptide motifs located within the RGG domain of Npl3p serve as the substrate docking motifs. However, a single docking motif is sufficient for its stable interaction with the kinase. Methylation of the docking motifs abolishes kinase binding and phosphorylation of Npl3p. Remarkably, removal of the docking groove in the kinase or the docking motifs of the substrate does not reduce the overall catalytic efficiency of the phosphorylation reaction in any significant manner. We suggest that docking interaction between Sky1p and Npl3p is essential for substrate recruitment and binding specificity.     

Nucleotide-induced conformational changes in the Saccharomyces cerevisiae SR protein kinase,Sky1p,revealed by X-ray crystallography

Conformational changes are thought to play a key role in the function of active protein kinases, although little is known about how these changes relate to the mechanism of phosphorylation. Here we present four high-resolution structures of a single crystal form of Sky1p, a constitutively active serine kinase implicated in yeast RNA processing, each in a different state of nucleotide binding. By comparing the apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformational changes caused by ATP binding or conversion from nucleotide reactant to product. Rotation of the small lobe of the kinase closes the cleft upon binding, allowing the nucleotide to interact with residues from both lobes of the kinase, although some interactions thought to be important for phosphotransfer are missing in the ATP-containing structure. In the apoenzyme, a kinase-conserved phosphate-anchoring loop is in a twisted conformation that is incompatible with ADP and ATP binding, providing a potential mechanism for facilitating ADP release in Sky1p. The nonhydrolyzable ATP analogue AMP-PNP binds in a unique mode that fails to induce lobe closure. This observation, along with comparisons between the two independent molecules in the asymmetric unit of each structure, has provided new molecular details about how the nucleotide binds and induces closure. Finally, we have used mutational analysis to establish the importance of a glycine within the linker that connects the two lobes of Sky1p.     

Regulating SR protein phosphorylation through regions outside the kinase domain of SRPK1

SR proteins (splicing factors containing arginine-serine repeats) are essential splicing factors whose phosphorylation by the SR-specific protein kinase (SRPK) family regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, nonhomologous spacer insert domain (SID) that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation. To investigate the conformation of the full-length kinase and the functional role of noncatalytic regions, we performed hydrogen-deuterium exchange and steady-state kinetic experiments on SRPK1. Unlike the kinase core, the large SID lacks stable, hydrogen-bonded structure and may provide an intrinsically disordered region for chaperone interactions. Conversely, the N-terminus, which positively regulates SR protein binding, adopts a stable structure when the insert domain is present and stabilizes a docking groove in the large lobe of the kinase domain. The N-terminus and SID equally enhance SR protein turnover by altering the stability of several catalytic loop segments. These studies reveal that SRPK1 uses an N-terminal extension and a large, intrinsically disordered region juxtaposed to a stable structure to facilitate high-affinity SR protein interactions and phosphorylation rates.     

Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation

Mammalian SR proteins are currently thought to function in mRNA export as well as splicing. They contain multiple phosphorylated serine/arginine (RS/SR) dipeptides. Although SR domains can be phosphorylated by many kinases in vitro, the physiologically relevant kinase(s), and the role(s) of these modifications in vivo have remained unclear. Npl3 is a shuttling protein in budding yeast that we showed previously to be a substrate for the mammalian SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1. Here we demonstrate that Sky1p phosphorylates only one of Npl3p's eight SR/RS dipeptides. Mutation of the C-terminal RS to RA, or deletion of SKY1, results in the cytoplasmic accumulation of Npl3p. The redistribution of Npl3p is accompanied by its increased association with poly(A)+ RNA and decreased association with its import receptor, Mtr10p, in vivo. We propose that phosphorylation of Npl3p by the cytoplasmically localized Sky1p is required for efficient release of mRNA upon termination of export.     

Protein kinase CK2 phosphorylates and activates the SR protein-specific kinase 1

The serine/arginine subfamily of protein kinases has been conserved throughout evolution and its members are thought to play important roles in the regulation of multiple cellular processes. Mammalian SRPK1 has been considered as a constitutively active kinase that is predominantly expressed in testis. In the present study, recombinant GST-SRPK1 was used as substrate to identify potential protein kinase(s) in testis extracts, involved in phosphorylating and thereby regulating the activity of this enzyme. Using a panel of chromatography media, inhibition by heparin, immunoblot analysis, and phosphopeptide mapping, CK2 was determined to be the major kinase that phosphorylates SRPK1. Phosphorylation of SRPK1 by CK2 occurred mainly at Ser(51) and Ser(555) in vitro, and resulted in approximately 6-fold activation of the enzyme. These findings suggest that SRPK1 may be an important cellular target for CK2 action.     

Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.     

Cellular localization and phosphorylation of Hrb1p is independent of Sky1p

Protein phosphorylation plays a major role in regulating cellular functions. We have previously demonstrated that Sky1p, the SR protein kinase of the budding yeast Saccharomyces cerevisiae, is a regulator of polyamine transport and ion homeostasis. Since its kinase activity was demonstrated essential for fulfilling these roles, we assumed that Sky1p function via substrates phosphorylation. Using an in vitro phosphorylation assay, we have identified Hrb1p as a putative Sky1p substrate. However, phosphorylation analysis in WT and sky1Delta cells and localization studies disproved Hrb1p as a true Sky1p substrate, although a segment of the RS domain is required for determining its subcellular localization. Furthermore, we demonstrate that Hrb1p and additional putative Sky1p substrates, identified by computational approach, are not involved in mediating the spermine tolerant phenotype of sky1Delta cells.     

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340)

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.     

Interplay between SRPK and Clk/Sty kinases in phosphorylation of the splicing factor ASF/SF2 is regulated by a docking motif in ASF/SF2

The arginine-serine (RS)-rich domain of the SR protein ASF/SF2 is phosphorylated by SR protein kinases (SRPKs) and Clk/Sty kinases. However, the mode of phosphorylation by these kinases and their coordination in the biological regulation of ASF/SF2 is unknown. Here, we report the crystal structure of an active fragment of human SRPK1 bound to a peptide derived from an SR protein. This structure led us to identify a docking motif in ASF/SF2. We find that this docking motif restricts phosphorylation of ASF/SF2 by SRPK1 to the N-terminal part of the RS domain - a property essential for its assembly into nuclear speckles. We further show that Clk/Sty causes release of ASF/SF2 from speckles by phosphorylating the C-terminal part of its RS domain. These results suggest that the docking motif of ASF/SF2 is a key regulatory element for sequential phosphorylation by SRPK1 and Clk/Sty and, thus, is essential for its subcellular localization.     

Exploring some of the physico-chemical properties of the LAMMER protein kinase DOA of Drosophila

Members of the highly conserved LAMMER family of protein kinases have been described in all eukaryotes. LAMMER kinases possess markedly similar peptide motifs in their kinase catalytic subdomains that are responsible for phosphotransfer and substrate interaction, suggesting that family members serve similar functions in widely diverged species. This hypothesis is supported by their phosphorylation of SR and SR-related proteins in diverged species. Here we describe a 3-dimensional homology model of the catalytic domain of DOA, a representative LAMMER kinase, encoded by the Drosophila locus Darkener of apricot (Doa). Homology modeling of DOA based on a Sky1p template revealed a highly conserved structural framework within conserved core regions. These adopt typical kinase folding like that of other protein kinases. However, in contrast to Sky1p, some structural features, such as those in helix ?C suggest that the DOA kinase is not a constitutively active enzyme but requires activation. This may occur by phosphorylation within an activation loop that forms a broad turn and in which interactions between the side chains occur across the loop. The fold of the activation loop is stabilized through interactions with residues in the C-terminal tail, which is not part of the conserved kinase core and is variable among protein kinases. Immediately following the activation loop in the segment between the ?9 sheet and helix ?F is a P+1 loop. The electrostatic surface potential of the DOA substrate binding groove is largely negative, as it is in other known SR protein kinases, suggesting that DOA substrates must be basic. All differences between D. melanogaster and other Drosophila species are single amino acid changes situated in regions outside of any ?-helices or ?-sheets, and after modeling these had absolutely no visible effect on protein structure. The absence of evolved amino acid changes among 12 Drosophila species that would cause at least predictable changes in DOA structure indicate that evolution has already selected evolved mutations for having minimal effect on kinase structure.     

SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development

SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development.