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

Abstract

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and ∼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. The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and ∼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. Pre-mRNA splicing and mRNA export are complex processes, which involve a large number of protein and RNA factors (1Hastings M.L. Krainer A.R. Curr. Opin. Cell Biol. 2001; 13: 302-309Crossref PubMed Scopus (392) Google Scholar, 2Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (2032) Google Scholar, 3Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar, 4Izaurralde E. Nat. Struct. Mol. Biol. 2004; 11: 210-212Crossref PubMed Scopus (17) Google Scholar). SR proteins, a class of non-small nuclear ribonucleoprotein splicing factors, participate in every step in the spliceosome assembly and catalysis (5Sanford J.R. Longman D. Caceres J.F. Prog. Mol. Subcell Biol. 2003; 31: 33-58Crossref PubMed Scopus (60) Google Scholar). SR proteins have unique domain architecture. The N-terminal domain contains one or two RNA recognition motifs (RRMs) 3The abbreviations used are: RRM, RNA recognition motif; SRPK, SR protein kinase 1; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; wt, wild type. 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 (6Fu X.D. RNA (N. Y.). 1995; 1: 663-680PubMed Google Scholar). 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 (6Fu X.D. RNA (N. Y.). 1995; 1: 663-680PubMed Google Scholar, 7Blencowe B.J. Bowman J.A. McCracken S. Rosonina E. Biochem. Cell Biol. 1999; 77: 277-291Crossref PubMed Scopus (107) Google Scholar, 8Colwill K. Feng L.L. Yeakley J.M. Gish G.D. Caceres J.F. Pawson T. Fu X.D. J. Biol. Chem. 1996; 271: 24569-24575Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Rossi F. Labourier E. Forne T. Divita G. Derancourt J. Riou J.F. Antoine E. Cathala G. Brunel C. Tazi J. Nature. 1996; 381: 80-82Crossref PubMed Scopus (289) Google Scholar, 10Stojdl D.F. Bell J.C. Biochem. Cell Biol. 1999; 77: 293-298Crossref PubMed Scopus (81) Google Scholar). 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 ∼10–12 of these serines in the N-terminal RS1 region of the RS domain (11Velazquez-Dones A. Hagopian J.C. Ma C.T. Zhong X.Y. Zhou H. Ghosh G. Fu X.D. Adams J.A. J. Biol. Chem. 2005; 280: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This multisite phosphorylation occurs in a processive manner where the kinase remains bound to the substrate until all sites are phosphorylated (12Aubol B.E. Chakrabarti S. Ngo J. Shaffer J. Nolen B. Fu X.D. Ghosh G. Adams J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12601-12606Crossref PubMed Scopus (91) Google Scholar). 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 Npl3p and Gbp2p (13Windgassen M. Krebber H. EMBO Rep. 2003; 4: 278-283Crossref PubMed Scopus (48) Google Scholar, 14Gilbert W. Siebel C.W. Guthrie C. RNA (N. Y.). 2001; 7: 302-313Crossref PubMed Scopus (121) Google Scholar, 15Yun C.Y. Fu X.D. J. Cell Biol. 2000; 150: 707-718Crossref PubMed Scopus (138) Google Scholar). However, unlike the mammalian SR proteins, the yeast proteins do not have a classic RS domain with long stretches of arginine/serine repeats (6Fu X.D. RNA (N. Y.). 1995; 1: 663-680PubMed Google Scholar, 16Dreyfuss G. Matunis M.J. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1362) Google Scholar) (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 phosphorylated by Sky1p (15Yun C.Y. Fu X.D. J. Cell Biol. 2000; 150: 707-718Crossref PubMed Scopus (138) Google Scholar). This modification is essential for the nuclear import of Npl3p, facilitated by an importin, Mtr10p (17Senger B. Simos G. Bischoff F.R. Podtelejnikov A. Mann M. Hurt E. EMBO J. 1998; 17: 2196-2207Crossref PubMed Scopus (159) Google Scholar). Defects in phosphorylation cause accumulation of Npl3p in the cytoplasm with enhanced mRNA binding activity (14Gilbert W. Siebel C.W. Guthrie C. RNA (N. Y.). 2001; 7: 302-313Crossref PubMed Scopus (121) Google Scholar, 15Yun C.Y. Fu X.D. J. Cell Biol. 2000; 150: 707-718Crossref PubMed Scopus (138) Google Scholar). Recent experiments reveal that the Gbp2p RS-repeat motif, which contains three successive RS-dipeptides, is critical for the nuclear import of Gbp2p (Fig. 1a) (13Windgassen M. Krebber H. EMBO Rep. 2003; 4: 278-283Crossref PubMed Scopus (48) Google Scholar). The domain arrangement in SRPKs is unique in that these kinases contain a large insert, bifurcating the kinase core domain (Fig. 1a). Although the insert region, known as the spacer domain, bears little sequence homology among SRPKs across species, its precise location with respect to the core kinase domain suggests that it may play a similar function in most SRPKs. Indeed, in both yeast and human kinases, the spacer domain plays a role in the subcellular localization of these kinases (18Ding J.H. Zhong X.Y. Hagopian J.C. Cruz M.M. Ghosh G. Feramisco J. Adams J.A. Fu X.D. Mol. Biol. Cell. 2006; 17: 876-885Crossref PubMed Scopus (95) Google Scholar). Both SRPK1 and Sky1p also have non-homologous extensions at their N termini. Sky1p has a 40-residue-long extension at its C terminus, whereas the C terminus of SRPK1 has no such extension (19Nolen B. Yun C.Y. Wong C.F. McCammon J.A. Fu X.D. Ghosh G. Nat. Struct. Biol. 2001; 8: 176-183Crossref PubMed Scopus (70) Google Scholar). The x-ray structure and subsequent deletion analysis of Sky1p reveal that its non-conserved C-terminal tail makes contact with the activation loop of the kinase. Deletion of only 10 residues from the C terminus of Sky1p drastically reduces the catalytic activity of Sky1p (19Nolen B. Yun C.Y. Wong C.F. McCammon J.A. Fu X.D. Ghosh G. Nat. Struct. Biol. 2001; 8: 176-183Crossref PubMed Scopus (70) Google Scholar). On the other hand, the entire spacer domain and N-terminal 137 amino acids of Sky1p can be removed without compromising its in vitro activity (19Nolen B. Yun C.Y. Wong C.F. McCammon J.A. Fu X.D. Ghosh G. Nat. Struct. Biol. 2001; 8: 176-183Crossref PubMed Scopus (70) Google Scholar). In SRPK1, both ends of the previously designated spacer domain fold into helical structures and interact with the kinase core. Deletion analyses of SRPK1 have revealed that, unlike Sky1p, removal of the designated spacer domain in its entirety results in reduced stability of SRPK1 (20Ngo J.C. Gullingsrud J. Giang K. Yeh M.J. Fu X.D. Adams J.A. McCammon J.A. Ghosh G. Structure. 2007; 15: 123-133Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Despite the critical differences in the primary and tertiary structures between SRPK1 and Sky1p, they can phosphorylate each other's substrate in vivo and in vitro (21Schenk P.W. Boersma A.W. Brandsma J.A. den Dulk H. Burger H. Stoter G. Brouwer J. Nooter K. Cancer Res. 2001; 61: 6982-6986PubMed Google Scholar, 22Siebel C.W. Feng L. Guthrie C. Fu X.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5440-5445Crossref PubMed Scopus (106) Google Scholar). SRPK1 also can functionally substitute for Sky1p in yeast suggesting that the basic mechanism of phosphorylation is evolutionarily conserved from yeast to humans. Perhaps it is not too surprising as the yeast substrate is phosphorylated only at a single site and that recognition of the phosphorylation site peptide of Npl3p is not different between these kinases. However, it is unclear if the yeast enzyme can phosphorylate the mammalian substrates at multiple sites in a processive manner. If so, this would imply the development of an evolutionarily conserved mechanism of phosphorylation among SRPKs. Our results show that, despite significant differences in the mechanism of how Sky1p and SRPK1 maintain their active states, these two kinases display an evolutionarily conserved mode of substrate binding and catalysis. We demonstrate here that catalysis and substrate recognition are conserved among SR protein kinases. However, only the SR protein substrates such as ASF/SF2 undergo processive phosphorylation but not the SR-like protein Gbp2p. Cloning and Expression of Recombinant Proteins—Expression and purification of His-SRPK1ΔNS1, His-Sky1pΔN(137)ΔS, mutants of Sky1p, SRPK1 K109M (trap), and Npl3p have been described previously. The cloning of ASF/SF2 in Escherichia coli expression vector has been described earlier (11Velazquez-Dones A. Hagopian J.C. Ma C.T. Zhong X.Y. Zhou H. Ghosh G. Fu X.D. Adams J.A. J. Biol. Chem. 2005; 280: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The expression vector was transformed into E. coli and in at at with and the was in of and The and the ASF/SF2. The was in of MOPS, The was an and with of was with of with 1 The protein was bound to binding for 1 at The was extensively by of in the with removal of was with 20 MOPS, and with a of The RGG domain Gbp2p residues was by a at amino in a vector Gbp2p as a Gbp2p mutants by single step Gbp2p was in E. coli to an and for at with The in of 1 DTT, 1 and and by The was for at and the was a The was with of and with 10 The protein was of 1 of Gbp2p In in the 10 1 1 DTT, and of at using and for 1 at a of and a and to as previously (11Velazquez-Dones A. Hagopian J.C. Ma C.T. Zhong X.Y. Zhou H. Ghosh G. Fu X.D. Adams J.A. J. Biol. Chem. 2005; 280: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). for of Npl3p and using of three in vitro kinase using of kinase and substrate as previously (11Velazquez-Dones A. Hagopian J.C. Ma C.T. Zhong X.Y. Zhou H. Ghosh G. Fu X.D. Adams J.A. J. Biol. Chem. 2005; 280: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The no SRPK1 K109M in which the kinase and substrate at for with The with the substrate at for the of active kinase and subsequent with The kinase and substrate with a of and to of and The mechanism of phosphorylation was by of the of the to that of the and processive and analyses as previously described (11Velazquez-Dones A. Hagopian J.C. Ma C.T. Zhong X.Y. Zhou H. Ghosh G. Fu X.D. Adams J.A. J. Biol. Chem. 2005; 280: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). of phosphorylated protein in 10 1 DTT, at with the of in a of 20 with three of DTT, experiments in two In the step proteins or in binding DTT, from 1 bound to and We 10 of protein to bound to In the kinase was to the bound to or or in of binding with of 10 was to each each was at for and a or a and to an with or from and Cruz Sky1p ASF/SF2 at has been previously shown that SRPK1 can functionally in vivo J.M. H. J. J.A. Fu X.D. J. Cell Biol. 1999; PubMed Scopus (121) Google Scholar). The of with SRPK1 that the mammalian kinase can phosphorylate Npl3p and other Sky1p substrates at a for The structures of Sky1p and SRPK1 shown (Fig. and a similar mechanism of substrate recognition by these two kinases J.C. Chakrabarti S. J.H. A. Nolen B. B.E. Adams J.A. Fu X.D. Ghosh G. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the substrates for these two kinases are different in of the number of phosphorylation This to how each of these kinases phosphorylates the heterologous in vitro kinase was to the ability of and to as Sky1p and SRPK1, to phosphorylate ASF/SF2 and Npl3p, experiments show that both Sky1p and SRPK1 phosphorylate ASF/SF2 (Fig. and Npl3p (Fig. results that the SRPKs may and phosphorylate these two substrates using similar further SRPK1, Sky1p also phosphorylates ASF/SF2 at multiple we have the of We used to the number of to ASF/SF2 by SRPK1 and In these ASF/SF2 was with a of Sky1p or SRPK1 for in the or of at and using (Fig. In the we that the ASF/SF2 1 and and Both to a for and for in the case of SRPK1 wt, with the of substrate (Fig. a in was for and for the of Sky1p, to the of substrate by Sky1p (Fig. of the with ASF/SF2 and in no further in not that an in the was a of that is of active ASF/SF2 Sky1p ASF/SF2 with tested the binding of ASF/SF2 for Sky1p and SRPK1 using a experiments showed that both SRPK1 and Sky1p (Fig. a and whereas bound Sky1p only (Fig. a and Although SRPK1 can phosphorylate Npl3p, to SRPK1 in where it with This suggests that binding of the complex is the The is a and not a of has been shown previously that SRPK1 ASF/SF2 with a of (12Aubol B.E. Chakrabarti S. Ngo J. Shaffer J. Nolen B. Fu X.D. Ghosh G. Adams J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12601-12606Crossref PubMed Scopus (91) Google Scholar). We to if Sky1p bind to ASF/SF2 with a similar a single at different kinase with a ASF/SF2 single at SRPK1 it was shown previously that ASF/SF2 phosphorylation is but at SRPK1 phosphorylation are (12Aubol B.E. Chakrabarti S. Ngo J. Shaffer J. Nolen B. Fu X.D. Ghosh G. Adams J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12601-12606Crossref PubMed Scopus (91) Google Scholar). The of the is to the of ASF/SF2 that a can be from a of bound enzyme the of the we Sky1p from to to at which the (Fig. We that Sky1p, the a single an However, at Sky1p, a in the mechanism was that, at Sky1p or most of the ASF/SF2 is bound by the enzyme, that the for Sky1p and ASF/SF2 is that Sky1p has a similar for ASF/SF2 as SRPK1. Sky1p further differences in the mechanism of ASF/SF2 phosphorylation by Sky1p, we used a as previously (11Velazquez-Dones A. Hagopian J.C. Ma C.T. Zhong X.Y. Zhou H. Ghosh G. Fu X.D. Adams J.A. J. Biol. Chem. 2005; 280: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In this the kinase and substrate are and the is with the of and to bind ASF/SF2 (Fig. we used a of SRPK1 K109M that well but phosphorylate ASF/SF2 (11Velazquez-Dones A. Hagopian J.C. Ma C.T. Zhong X.Y. Zhou H. Ghosh G. Fu X.D. Adams J.A. J. Biol. Chem. 2005; 280: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). If the is no ASF/SF2 is as the the the (Fig. However, if the is one or of ASF/SF2, from the active site of Sky1p, be by K109M in We used ASF/SF2, or Sky1p, and for the (Fig. and We that, at Sky1p the is is a in phosphorylation of ASF/SF2 in the between two different of In the ASF/SF2 is with the active Perhaps the is to the kinase at However, these show that, similar to SRPK1, Sky1p is a processive Sky1p and SRPK1 a to have previously shown that the SRPK1 docking groove is for binding and processive phosphorylation of ASF/SF2 J.C. Chakrabarti S. J.H. A. Nolen B. B.E. Adams J.A. Fu X.D. Ghosh G. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We have also shown that Sky1p contains a docking groove Nolen B. Adams J.A. Ghosh G. J. Mol. Biol. 2007; PubMed Scopus Google Scholar). within the docking we showed that the docking groove in Sky1p plays an role for Npl3p phosphorylation. we to if this same docking groove of Sky1p is also for ASF/SF2 We tested the binding between Sky1p docking groove mutants and and using of these mutants contains amino to and and and Both of in the docking groove and drastically reduced the binding of ASF/SF2 with Sky1p (Fig. In results that both yeast and human SRPKs to ASF/SF2, and the kinase docking groove is to their Our results also imply that the docking motif interaction plays a role in substrate phosphorylation. a in Phosphorylation of tested the of the Sky1p docking groove and processive phosphorylation of ASF/SF2 using the as described earlier (Fig. and We used ASF/SF2, of the kinases, and for the We that ASF/SF2 phosphorylation by Sky1p is not by the of (Fig. Our results demonstrate that Sky1p processively phosphorylates ASF/SF2, whereas only (Fig. are with that the Sky1p not phosphorylation of Npl3p, whereas the Sky1p Nolen B. Adams J.A. Ghosh G. J. Mol. Biol. 2007; PubMed Scopus Google Scholar). However, these results to be with experiments that showed that both mutants in ASF/SF2 is to the of the kinases for ASF/SF2 to each other using a Although we that both Sky1p mutants are to bind ASF/SF2 in a it is that the has a ASF/SF2 the is that we used a protein the that is the of the of both appears that the of the complex is for the phosphorylation to Sky1p which suggests that, is this interacts with the substrate to an that processive activity also be by the and the of may be for Sky1p with Gbp2p and at ability of Sky1p to processive phosphorylation of heterologous substrates to for yeast substrates that be processively phosphorylated at multiple protein contains an domain Npl3p where three serines are present within a of RS dipeptides (Fig. 1a). Gbp2p also contains an RGG domain C-terminal to the RS In we have shown that the docking groove of Sky1p interacts with the RGG domain of Npl3p, and this docking interaction is for phosphorylation of Npl3p Nolen B. Adams J.A. Ghosh G. J. Mol. Biol. 2007; PubMed Scopus Google Scholar). We also showed that the RGG domain of Npl3p makes with an docking groove in Sky1p Nolen B. Adams J.A. Ghosh G. J. Mol. Biol. 2007; PubMed Scopus Google Scholar). The of an RS motif in the N terminus to with an RGG motif in Gbp2p suggests that Sky1p may also similar docking with the RGG domain to phosphorylate the serines in the RS if Sky1p interacts with we have the domain of Gbp2p as a protein (Fig. Gbp2p is an protein, and to this protein to We tested its binding to Sky1p and the docking groove mutants and using experiments (Fig. in where two different of these kinases and 10 We Sky1p and bind to but the bound We also that SRPK1 also bind Gbp2p. these results that Sky1p utilizes a similar binding mechanism to interact with Gbp2p and if Sky1p phosphorylates these serines or other serines within the domain, we all three serines in the RS region at and of to In vitro phosphorylation of the by Sky1p was reduced with (Fig. if multisite phosphorylation occurs in we kinase with three single mutants where each of these serines is to (Fig. The kinase results demonstrated that Sky1p phosphorylates Gbp2p at multiple sites between and We further a which also phosphorylation by Sky1p not Our suggests that Sky1p this RS at the in Npl3p was previously shown to be for Npl3p phosphorylation (19Nolen B. Yun C.Y. Wong C.F. McCammon J.A. Fu X.D. Ghosh G. Nat. Struct. Biol. 2001; 8: 176-183Crossref PubMed Scopus (70) Google Scholar). at as a with respect to We this to and the revealed no Gbp2p phosphorylation. we if Sky1p processively phosphorylates these serines using the (Fig. We not phosphorylation of Gbp2p that of the at a of of the In the of of of the it is to the mechanism of phosphorylation. The of phosphorylation of Gbp2p suggests that multisite phosphorylation of this substrate may not in a processive manner. In this we have demonstrated that, SRPK1, Sky1p phosphorylates ASF/SF2 at multiple SRPK1 phosphorylates ASF/SF2 at an of whereas Sky1p phosphorylates similar of phosphorylation by both kinases that serines of ASF/SF2 also be phosphorylated by these kinases. we showed that both SRPK1 and Sky1p contain a peptide docking groove the of the large of the kinase by an insert unique to the protein and the loop and J.C. Chakrabarti S. J.H. A. Nolen B. B.E. Adams J.A. Fu X.D. Ghosh G. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The x-ray structures of Sky1p and SRPK1 bound to an peptide revealed that the kinase docking groove the peptide in a manner for many other where the peptide a groove in the kinase it in both and Both Sky1p and SRPK1 their docking to interact with their Npl3p and ASF/SF2. We show here that Sky1p ASF/SF2 with and in the Sky1p docking groove ASF/SF2 results shown here a conserved mechanism of ASF/SF2 recognition and phosphorylation by both SRPK1 and the conserved docking in SRPK1 and Sky1p, the docking motifs of their substrates also conserved The docking motif of ASF/SF2 between the and the RS domain is rich in basic the docking motif of Npl3p, the entire RGG domain, is also rich in basic residues Nolen B. Adams J.A. Ghosh G. J. Mol. Biol. 2007; PubMed Scopus Google Scholar). basic residues are the for an interaction with conserved residues in the kinase docking to substrate In the case of the phosphorylation sites are in the N-terminal domain, whereas an RGG region the docking motif of Npl3p is C-terminal to the phosphorylation Although the physical of the phosphorylation site and the docking motif are in Npl3p and their recognition by Sky1p is by of the docking groove in in experiments suggests that Sky1p Npl3p and Gbp2p We have demonstrated that Sky1p the ability to processively phosphorylate a heterologous SR protein This the if there are yeast substrates that also undergo processive phosphorylation. The most of processive phosphorylation is which we have demonstrated to be phosphorylated multiple by However, experiments a mechanism that is to be We not the of the However, it is to that ASF/SF2, was to both Sky1p and SRPK1 at We used kinase and which is the used in the experiments and well that of the of the In this phosphorylation was This of phosphorylation with the suggests that phosphorylation of Gbp2p by Sky1p is most We can at that the mechanism of phosphorylation of SR and SR-like substrates by SRPKs is The results an of processive phosphorylation. from and experiments a complex In to the ability of a substrate multiple phosphorylation sites and to bind the kinase with other be Perhaps the interaction between the kinase and substrate at the docking and active sites is essential for processive phosphorylation. is to that the of ASF/SF2 is also in the binding with SRPK1 and with Sky1p as In the of Npl3p are not in binding this it is that and of these the domain be for processive phosphorylation. Thus, all RGG proteins such as the protein J. 1996; PubMed Google Scholar, 1998; 12: PubMed Scopus Google Scholar, EMBO J. 2003; PubMed Scopus Google Scholar) with multiple phosphorylation sites may undergo phosphorylation. is to this a role for processive phosphorylation in has to be We that processive phosphorylation is phosphorylation, phosphorylation the substrates to be in their in a manner in the are in for their In yeast, the of splicing may not these proteins to function in such a manner. We Chakrabarti for of the We also Ma for the