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Casein Kinase I Associates with Members of the Centaurin-α Family of Phosphatidylinositol 3,4,5-Trisphosphate-binding Proteins*

Open AccessPublished:June 01, 2001DOI:https://doi.org/10.1074/jbc.M010005200
      Mammalian casein kinases I (CKI) belong to a family of serine/threonine protein kinases involved in diverse cellular processes including cell cycle progression, membrane trafficking, circadian rhythms, and Wnt signaling. Here we show that CKIα co-purifies with centaurin-α1 in brain and that they interact in vitro and form a complex in cells. In addition, we show that the association is direct and occurs through the kinase domain of CKI within a loop comprising residues 217–233. These residues are well conserved in all members of the CKI family, and we show that centaurin-α1 associates in vitrowith all mammalian CKI isoforms. To date, CKIα represents the first protein partner identified for centaurin-α1. However, our data suggest that centaurin-α1 is not a substrate for CKIα and has no effect on CKIα activity. Centaurin-α1has been identified as a phosphatidylinositol 3,4,5-trisphosphate-binding protein. Centaurin-α1contains a cysteine-rich domain that is shared by members of a newly identified family of ADP-ribosylation factor guanosine trisphosphatase-activating proteins. These proteins are involved in membrane trafficking and actin cytoskeleton rearrangement, thus supporting a role for CKIα in these biological events.
      CKI(s)
      casein kinase(s) I
      PtdIns (3
      4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
      ARF
      ADP-ribosylation factor
      GAP
      guanosine trisphosphatase-activating protein
      PH
      pleckstrin homology
      HA
      hemagglutinin
      GST
      glutathione S-transferase
      PAGE
      polyacrylamide gel electrophoresis
      DTT
      dithiothreitol
      The casein kinase I (CKI)1 family of serine/threonine kinases is ubiquitously expressed in a range of eukaryotes including yeast and humans as well as in plants (reviewed in Ref.
      • Gross S.D.
      • Anderson R.A.
      ). Seven isoforms from distinct genes are expressed in mammals (CKI α, μ, γ1, γ2, γ3, δ, and ε), four inSaccharomyces cerevisiae (Hrr25, Yck1, Yck2, and Yck3), and five in Schizosaccharomyces pombe (Cki1, Cki2, Cki3, Hhp1, and Hhp2). The CKI family is characterized by a conserved core kinase domain and variable amino- and carboxyl-terminal tails.
      Yeast CKI isoforms are involved in DNA repair (
      • Hoekstra M.F.
      • Liskay R.M.
      • Ou A.C.
      • DeMaggio A.J.
      • Burbee D.G.
      • Heffron F.
      ,
      • Dhillon N.
      • Hoekstra M.F.
      ,
      • Ho U.
      • Mason S.
      • Kobayashi R.
      • Hoekstra M.
      • Andrews B.
      ). Recently, many reports (
      • Robinson L.C.
      • Menold M.M.
      • Garret S.
      • Culbertson M.R.
      ,
      • Robinson L.C.
      • Bradley C.
      • Bryan J.D.
      • Jerome A.
      • Kweon Y.
      • Panek H.R.
      ,
      • Wang X.
      • Hoekstra M.F.
      • DeMaggio A.J.
      • Dhillon N.
      • Vancura A.
      • Kuret J.
      • Johnston G.C.
      • Singer R.A.
      ,
      • Panek H.R.
      • Stepp J.D.
      • Engle H.M.
      • Marks K.M.
      • Tan P.K.
      • Lemmon S.K.
      • Robinson L.C.
      ,
      • Hicke L.
      • Zanolari B.
      • Riezman H.
      ,
      • Friant S.
      • Zanolari B.
      • Riezman H.
      ,
      • Feng Y.
      • Davies N.G.
      ,
      • Marchal C.
      • Haguenaur-Tsapis R.
      • Urban-Grimal D.
      ) indicate that they also play a role in cyaff8inesis and in vesicle trafficking especially in endocytosis. The functions of the mammalian isoforms are less well understood, but based on high homology with their yeast counterparts, they may have similar biological functions. CKIε and CKIδ play a role in the regulation of p53 (
      • Knippschild U.
      • Milne D.M.
      • Campbell L.E.
      • De Maggio A.J.
      • Christenson E.
      • Hoekstra M.F.
      • Meek D.W.
      ,
      • Dumaz N.
      • Milne D.M.
      • Meek D.W.
      ). CKIε has also been implicated in circadian rhythms inDrosophila (
      • Kloss B.
      • Price J.L.
      • Saez L.
      • Blau J.
      • Rothenfluh A.
      • Wesley C.S.
      • Young M.W.
      ,
      • Lowrey P.L.
      • Shimomura K.
      • Anaff2h M.P.
      • Yamazaki S.
      • Zemenides P.D.
      • Ralph M.R.
      • Menaker M.
      • Takahashi J.S.
      ) and in development by transducing the Wnt pathway (
      • Peters J.M.
      • McKay R.M.
      • McKay J.P.
      • Graff J.M.
      ,
      • Sakanaka C.
      • Leong P.
      • Xu L.
      • Harrison S.D.
      • Williams L.T.
      ). CKIγ might play a role in cyaff8inesis and/or in membrane trafficking (
      • Zhai L.
      • Graves P.R.
      • Robinson L.C.
      • Italiano M.
      • Culbertson M.R.
      • Rowles J.
      • Cobb M.H.
      • DePaoli-Roach A.A.
      • Roach P.J.
      ). CKIα has been shown to play a role in cell cycle progression (
      • Gross S.D.
      • Simerly C.
      • Schatten G.
      • Anderson R.A.
      ) and in membrane trafficking (
      • Gross S.D.
      • Hoffman D.P.
      • Fisette P.L.
      • Bass P.
      • Anderson R.A.
      ,
      • Faundez V.V.
      • Kelly R.B.
      ). Recently, CKIs have been shown to be implicated in regulating the nucleocytoplasmic localization of some substrates (
      • Zhu J.
      • Shibasaki F.
      • Price R.
      • Guillemot J.-C.
      • Yano T.
      • Dotsch V.
      • Wagner G.
      • Ferrara P.
      • McKeon F.
      ,
      • Vielhaber E.
      • Eide E.
      • Rivers A.
      • Gao Z.-H.
      • Virshup D.M.
      ).
      Several substrates, including nuclear and cytosolic proteins and membrane receptors, have been reported to be phosphorylated at leastin vitro by a CKI activity (reviewed in Ref.
      • Gross S.D.
      • Anderson R.A.
      ). CKI isoforms are thought to be constitutively active and second messenger-independent. However, it has been shown that CKIδ and CKIε are regulated by autophosphorylation (
      • Graves P.R.
      • Roach P.J.
      ,
      • Cegielska A.
      • Gietzen K.F.
      • Rivers A.
      • Virshup D.M.
      ,
      • Rivers A.
      • Gietzen K.F.
      • Vielhaber E.
      • Virshup D.M.
      ,
      • Fish Gietzen K.
      • Virshup D.M.
      ). CKIα is also autophosphorylated, but whether this has an effect on its activity is not well defined. CKIα is negatively regulated by PtdIns(4,5)P2 (
      • Gross S.D.
      • Hoffman D.P.
      • Fisette P.L.
      • Bass P.
      • Anderson R.A.
      ). Moreover, CKI isoforms have been reported to phosphorylate some of their substrates only if they were previously phosphorylated by another kinase two or three residues carboxyl-terminal to the CKI phosphorylation site. In this way, the effect of CKI is dependent on other kinases. CKIα is present in cells in different spliced forms (
      • Gross S.D.
      • Anderson R.A.
      ,
      • Fu Z.
      • Green C.L.
      • Bennett G.S.
      ) exhibiting different substrate specificities and differences in their protein-protein interactions.
      Although the yeast CKI isoforms have been well characterized, the functions of the mammalian CKI isoforms are much less known. Therefore, the identification of mammalian CKI substrates and CKI-binding proteins should help to clarify their cellular function(s). CKIα interacts with NF-AT4 (
      • Zhu J.
      • Shibasaki F.
      • Price R.
      • Guillemot J.-C.
      • Yano T.
      • Dotsch V.
      • Wagner G.
      • Ferrara P.
      • McKeon F.
      ), the paired helical filaments (
      • Kuret J.
      • Johnson G.S.
      • Cha D.
      • Christenson E.R.
      • DeMaggio A.J.
      • Hoekstra M.F.
      ), G-protein-coupled receptors (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ), and the AP-3 complex (
      • Faundez V.V.
      • Kelly R.B.
      ). CKIα also forms a complex with certain splicing factors but these interactions may be indirect (
      • Gross S.D.
      • Loijens J.C.
      • Anderson R.A.
      ).
      In the present study, we have shown that CKIα interacts with centaurin-α1. Centaurin-α1 is a PtdIns(3,4,5)P3-binding protein containing two PH domains (
      • Hammonds-Odie L.P.
      • Jackson T.R.
      • Profit A.A.
      • Blader I.J.
      • Turck C.W.
      • Prestwich G.D.
      • Theibert A.B.
      ,
      • Stricker R.
      • Hulser E.
      • Fischer J.
      • Jarchau T.
      • Walter U.
      • Lottspeich F.
      • Reiser G.
      ,
      • Tanaka K.
      • Imajoh-Ohmi S.
      • Sawada T.
      • Shirai R.
      • Hashimoto Y.
      • Iwasaki S.
      • Kaibuchi K.
      • Kanaho Y.
      • Terada Y.
      • Kimura K.
      • Nagata S.
      • Fukui Y.
      ) and a zinc finger motif similar to the one found in a newly identified family of ADP-ribosylation factor (ARF) guanosine trisphosphatase-activating proteins (GAP) (reviewed in Refs.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Donaldson J.G.
      ,
      • Donaldson J.G.
      • Jackson C.L.
      ,
      • Jackson T.R.
      • Keans B.G.
      • Theibert A.B.
      ). The yeast protein that shows the highest homology to centaurin-α1, Gcs1, also contains a zinc finger motif that confers its ARF-GAP activity (
      • Poon P.P.
      • Wang X.
      • Rotman M.
      • Huber I.
      • Cukierman E.
      • Cassel D.
      • Singer R.A.
      • Johnston G.C.
      ). Members of this family are involved in membrane trafficking and in actin cytoskeleton rearrangement. Our results suggest that CKIα plays a role in membrane trafficking and/or actin cytoskeleton rearrangement, thus confirming previous reports (
      • Gross S.D.
      • Hoffman D.P.
      • Fisette P.L.
      • Bass P.
      • Anderson R.A.
      ,
      • Faundez V.V.
      • Kelly R.B.
      ).

      DISCUSSION

      In this report we have identified centaurin-α1 as a novel CKIα partner based on the following evidence: (a) they co-purified from brain after elution from four chromaaff5raphy steps; (b) centaurin-α1 associates in vitro with CKIα indicating that the binding is direct; (c) the binding is specific as centaurin-α1does not interact in vitro with 14-3-3 ζ under the same conditions; (d) they form a protein complex in COS-7 cells as shown in immunoprecipitation experiments; (e) centaurin-α1 interacts with residues 217–233 of CKIα using deletion mutants of CKIα; and (f) centaurin-α1 elutes from a peptide affinity chromaaff5raphy column containing residues 214–233 of CKIα.
      CKI isoforms are characterized by a conserved core kinase domain and by variable amino- and carboxyl-terminal tails. We report here that centaurin-α1 interacts with the kinase domain and not with the unique tails of CKIα. Moreover, a mutant of CKIδ deleted of its carboxyl-terminal domain binds to centaurin-α1 as well as does CKIδ, suggesting that the kinase domain of CKIδ represents the centaurin-binding site. In addition, the site of interaction within the kinase domain (residues 217–233) is present in a loop between two helices which has been proposed to represent an interaction domain for CKI targets (
      • Longenecker K.L.
      • Roach P.J.
      • Hurley T.D.
      ). The residues within that loop are well conserved among the CKI family. Indeed, we have shown that all mammalian CKI isoforms are able to associate with centaurin-α1 in vitro. This suggests that the same loop, present in all CKI isoforms, is responsible for the interaction with centaurin-α1.
      Centaurin-α1 and centaurin-α have been identified as PtdIns(3,4,5)P3-binding proteins (
      • Hammonds-Odie L.P.
      • Jackson T.R.
      • Profit A.A.
      • Blader I.J.
      • Turck C.W.
      • Prestwich G.D.
      • Theibert A.B.
      ,
      • Stricker R.
      • Hulser E.
      • Fischer J.
      • Jarchau T.
      • Walter U.
      • Lottspeich F.
      • Reiser G.
      ,
      • Tanaka K.
      • Imajoh-Ohmi S.
      • Sawada T.
      • Shirai R.
      • Hashimoto Y.
      • Iwasaki S.
      • Kaibuchi K.
      • Kanaho Y.
      • Terada Y.
      • Kimura K.
      • Nagata S.
      • Fukui Y.
      ). Phosphatidylinositol 3-kinase is mainly responsible for the synthesis of PtdIns(3,4,5)P3 by phosphorylating PtdIns(4,5)P2 at the 3-OH position (
      • Rameh L.E.
      • Cantley L.C.
      ). Phosphatidylinositol 3-kinase is involved in regulating various biological processes including membrane ruffling, membrane trafficking, and actin cytoskeleton regulation (
      • Martin T.F.J.
      ,
      • Corvera S.
      • D'Arrigo A.
      • Stenmark H.
      ,
      • Cullen P.J.
      • Venkateswarlu K.
      ). It is interesting to note that CKIα has recently been shown to interact with the clathrin adaptor AP-3 (
      • Faundez V.V.
      • Kelly R.B.
      ), another PtdIns(3,4,5)P3-binding protein (
      • Hao W.
      • Tan Z.
      • Prasad K.
      • Reddy K.K.
      • Chen L.
      • Prestwich G.D.
      • Falck J.R.
      • Shears S.B.
      • Lafer E.M.
      ). PtdIns(4,5)P2 has been shown to inhibit CKIα activity in vitro (
      • Gross S.D.
      • Hoffman D.P.
      • Fisette P.L.
      • Bass P.
      • Anderson R.A.
      ). However, the physiological relevance of the inhibition of CKIα by these two phospholipids remains to be demonstrated. Another link between CKI and the phosphoinositide pathway has been reported in S. pombe. The authors showed that a yeast CKI homologue, Cki1, phosphorylates and inhibits PtdIns(4)P 5-kinase (
      • Vancurova I.
      • Choi J.H.
      • Lin H.
      • Kuter J.
      • Vancura A.
      ).
      Centaurin-α1 belongs to a newly identified family of ARF-GAP proteins (reviewed in Refs.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Donaldson J.G.
      ,
      • Donaldson J.G.
      • Jackson C.L.
      ,
      • Jackson T.R.
      • Keans B.G.
      • Theibert A.B.
      ). Members of this family share a cysteine-rich GAP domain and contain several other domains such as PH domains, SH3 domains, and ankyrin repeats. These proteins are involved in vesicle trafficking and in actin cytoskeleton rearrangement. Therefore, our data support a role for CKIα in these biological events, in agreement with previous reports (
      • Gross S.D.
      • Hoffman D.P.
      • Fisette P.L.
      • Bass P.
      • Anderson R.A.
      ,
      • Faundez V.V.
      • Kelly R.B.
      ). Indeed, CKIα interacts with and phosphorylates the clathrin adaptor AP-3 (
      • Faundez V.V.
      • Kelly R.B.
      ), which is involved in endocytosis. It is interesting to note that a genetic interaction between yeast CKI and AP-3 was identified previously (
      • Panek H.R.
      • Stepp J.D.
      • Engle H.M.
      • Marks K.M.
      • Tan P.K.
      • Lemmon S.K.
      • Robinson L.C.
      ). Moreover, CKIα has been found to co-localize in neurones with synaptic vesicle markers and phosphorylates some vesicle synaptic associated proteins (
      • Gross S.D.
      • Hoffman D.P.
      • Fisette P.L.
      • Bass P.
      • Anderson R.A.
      ). Interestingly, centaurin-α1 has been shown to associate with presynaptic vesicular structures (
      • Kreutz M.R.
      • Bockers T.M.
      • Sabel B.A.
      • Hulser E.
      • Stricker R.
      • Reiser G.
      ). An actin-associated protein kinase shown to be a member of the CKI family phosphorylates actin in vitro(
      • Karino A.
      • Okano M.
      • Hatomi M.
      • Nakamura T.
      • Ohtsuki K.
      ). The molecular mass of the kinase (37 kDa) suggests that it could be CKIα, and we have shown that recombinant CKIα indeed phosphorylates actin.
      T. Dubois, S. K. Maciver, and A. Aitken, unpublished data.
      In addition, the protein DAH (Discontinuous Actin Hexagon) that interacts with the actin cytoskeleton has been shown to be phosphorylated by CKIin vitro (
      • Zhang C.X.
      • Rothwell W.F.
      • Sullivan W.
      • Hsieh T.-S.
      ).
      Members of the ARF-GAP family contain several domains for protein-protein interactions, and they have been shown to associate with a number of different proteins. This suggests that ARF-GAP proteins may act as scaffold proteins in addition to their function as GAP proteins. Whether other ARF-GAP proteins interact with CKI is not known. The ARF-GAP proteins Git1 and Git2 have been reported to regulate the internalization of some G-protein-coupled receptors (
      • Premont R.T.
      • Claing G.
      • Vitale N.
      • Freeman J.L.
      • Pitcher J.A.
      • Patton W.A.
      • Moss J.
      • Vaughan M.
      • Lefkowitz R.J.
      ,
      • Claing A.
      • Perry S.J.
      • Achiriloaie M.
      • Walker J.K.L.
      • Albanesi J.P.
      • Lefkowitz R.J.
      • Premont R.T.
      ,
      • Premont R.T.
      • Claing A.
      • Vitale N.
      • Perry S.J.
      • Lefkowitz R.J.
      ). CKIα has been shown to interact with and phosphorylate these G-protein-coupled receptors (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ,
      • Tobin A.B.
      • Totty N.F.
      • Sterlin A.E.
      • Nahorski S.R.
      ). In addition, most of the identified ARF-GAP proteins are involved in the Pak signaling pathway (reviewed in Ref.
      • Bagrodia S.
      • Cerione R.A.
      ). Intriguingly, CKIγ2 has been found to interact with the adaptor molecule Nck (
      • Lussier G.
      • Larose L.
      ) in a complex with Pak1 (
      • Voisin L.
      • Larose L.
      • Meloche S.
      ), thus raising the possibility that CKI may associate with other ARF-GAP proteins. Therefore, it would be important to investigate whether ARF-GAP proteins interact with CKI isoforms.
      Gcs1, the budding yeast homologue of centaurin-α1, also contains a cysteine-rich domain that is necessary for its ARF-GAP activity (
      • Poon P.P.
      • Wang X.
      • Rotman M.
      • Huber I.
      • Cukierman E.
      • Cassel D.
      • Singer R.A.
      • Johnston G.C.
      ). As yet, no ARF-GAP activity has been reported for centaurin-α1, but it is able of rescuing a ΔGcs1 strain mutant indicating that centaurin-α1 and Gcs1 may have similar function(s) (
      • Venkateswarlu K.
      • Oatey P.B.
      • Tavare J.M.
      • Jackson T.R.
      • Cullen P.J.
      ). Gcs1 has been shown to be necessary for the resumption of cell proliferation from stationary phase (
      • Ireland L.S.
      • Johnston G.C.
      • Drebot M.A.
      • Dhillon N.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Singer R.A.
      ) and is involved in endocytosis (
      • Wang X.
      • Hoekstra M.F.
      • DeMaggio A.J.
      • Dhillon N.
      • Vancura A.
      • Kuret J.
      • Johnston G.C.
      • Singer R.A.
      ). Gcs1 also plays a role in actin cytoskeleton regulation in vivo and binds to actin in vitro (
      • Blader I.J.
      • Jamie M.
      • Cope T.V.
      • Jackson T.R.
      • Profit A.A.
      • Greenwood A.F.
      • Drubin D.G.
      • Prestwich G.D.
      • Theibert A.B.
      ). As vesicle trafficking is closely associated to actin organization in yeast, Gcs1 may link vesicle trafficking and the actin cytoskeleton (
      • Blader I.J.
      • Jamie M.
      • Cope T.V.
      • Jackson T.R.
      • Profit A.A.
      • Greenwood A.F.
      • Drubin D.G.
      • Prestwich G.D.
      • Theibert A.B.
      ). Yeast CKIs (Yck1 and Yck2) were shown to suppress the Gcs1 blockage effects on cell proliferation and endocytosis (
      • Wang X.
      • Hoekstra M.F.
      • DeMaggio A.J.
      • Dhillon N.
      • Vancura A.
      • Kuret J.
      • Johnston G.C.
      • Singer R.A.
      ). The membrane association of Yck2 was necessary for this effect (
      • Wang X.
      • Hoekstra M.F.
      • DeMaggio A.J.
      • Dhillon N.
      • Vancura A.
      • Kuret J.
      • Johnston G.C.
      • Singer R.A.
      ). Yck1/2 is involved in cyaff8inesis, in bud development (
      • Robinson L.C.
      • Menold M.M.
      • Garret S.
      • Culbertson M.R.
      ,
      • Robinson L.C.
      • Bradley C.
      • Bryan J.D.
      • Jerome A.
      • Kweon Y.
      • Panek H.R.
      ), and regulation of the actin cytoskeleton as yckts mutants fail to depolarize the actin cytoskeleton during mitosis (
      • Robinson L.C.
      • Menold M.M.
      • Garret S.
      • Culbertson M.R.
      ). Another link between Gcs1 and CKI is the ankyrin repeat protein Akr1p. Gcs1 has been shown to interact with Akr1p in yeast two-hybrid experiments (
      • Kao L.R.
      • Peterson J.
      • Ji R.
      • Bender L.
      • Bender A.
      ). Akr1p and Yck1/2 regulate yeast endocytosis, and Akr1p regulates the plasma membrane localization of Yck1/2 (
      • Feng Y.
      • Davies N.G.
      ). These authors proposed that the Yck1/2 membrane localization may involve other proteins such as Gcs1 (
      • Kao L.R.
      • Peterson J.
      • Ji R.
      • Bender L.
      • Bender A.
      ).
      Our data suggest that CKIα does not phosphorylate centaurin-α and centaurin-α1. In addition centaurin-α1 has no effect on CKI activity. Therefore, what is the functional relevance of the interaction between CKIα and these PtdIns(3,4,5)P3-binding proteins? As CKIα does not contain a lipid binding domain, it may associate with membranes through interaction with other proteins. Centaurin-α1 may represent one of these proteins, as has been proposed for its yeast counterpart (see above and Ref.
      • Kao L.R.
      • Peterson J.
      • Ji R.
      • Bender L.
      • Bender A.
      ). CKIα may also represent a downstream target for centaurin-α1 as suggested by the results in budding yeast showing that CKIs suppress Gcs1 mutant phenotypes.
      In conclusion, we have shown an interaction between CKIα and centaurin-α1, a member of the ARF-GAP protein family that is involved in membrane trafficking and actin cytoskeleton regulation. Our present results are in agreement with data reported previously suggesting a role for CKI in membrane trafficking and/or regulation of the actin cytoskeleton. Our findings are further supported by evidence of a genetic link between CKI and Gcs1 in budding yeast.

      Acknowledgments

      We thank Frank McKeon (Department of Cell Biology, Harvard Medical School, Boston) and David Virshup (Department of Oncological Sciences, University of Utah, Salt Lake City, UT) for the CKIα(D136N) and CKIε (pKF182) plasmids, respectively. We are also grateful to Alex Peden for critical reading of this manuscript. We also thank C. Hyde for CKI peptide synthesis.

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