CKIα Is Associated with and Phosphorylates Star-PAP and Is Also Required for Expression of Select Star-PAP Target Messenger RNAs*

We have recently identified Star-PAP, a nuclear poly(A) polymerase that associates with phosphatidylinositol-4-phosphate 5-kinase Iα (PIPKIα) and is required for the expression of a specific subset of mRNAs. Star-PAP activity is directly modulated by the PIPKIα product phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), linking nuclear phosphoinositide signaling to gene expression. Here, we show that PI-4,5-P2-dependent protein kinase activity is also a part of the Star-PAP protein complex. We identify the PI-4,5-P2-sensitive casein kinase Iα (CKIα) as a protein kinase responsible for this activity and further show that CKIα is capable of directly phosphorylating Star-PAP. Both CKIα and PIPKIα are required for the synthesis of some but not all Star-PAP target mRNA, and like Star-PAP, CKIα is associated with these messages in vivo. Taken together, these data indicate that CKIα, PIPKIα, and Star-PAP function together to modulate the production of specific Star-PAP messages. The Star-PAP complex therefore represents a location where multiple signaling pathways converge to regulate the expression of specific mRNAs.

ical PAPs, the Star-PAP protein complex and architecture differ significantly from PAP␣, and consequently, Star-PAP specifically targets a select subset of mRNAs. This suggests that Star-PAP is a hybrid PAP that is required for the 3Ј-end formation of newly transcribed pre-mRNAs but functions in a regulatory role to control mRNA expression levels. Star-PAP has a unique domain structure relative to all other known PAPs (6). One unique feature is a 205-amino acid proline-rich region (PRR) inserted into the catalytic PAP core. The PRR splits the catalytic PAP domain, and this region represents a potential site for regulation of Star-PAP function.
Phosphoinositides, including PI-4,5-P 2 , are lipid signaling molecules that act as important regulators of numerous cellular functions (10). Phosphoinositides are generated ubiquitously in cells by multiple families of phosphoinositide kinases, which include PIPKI␣ (11). Phosphoinositide signaling specificity is based on the spatial restriction of these kinases to specific subcellular locations (12). This is often achieved through specific interactions between the phosphoinositide-generating enzymes and targeting factors, which are themselves often PI-4,5-P 2 -regulated proteins (13,14). This allows for the generation of inositol lipid second messengers at specific sites within the cell that, in turn, allows for precise targeting of individual phosphoinositide-sensitive pathways (12).
In the nucleus, PI-4,5-P 2 and PIPKI␣, as well as Star-PAP, are found in structures called nuclear speckles (6,15), which are nuclear bodies enriched in factors required for the processing of pre-mRNA (16). The direct interaction between Star-PAP and PIPKI␣ and the proximity of Star-PAP to PI-4,5-P 2 generation in nuclear speckles suggest that nuclear phosphoinositides are spatially positioned to modulate Star-PAP activity in vivo. The presence of PIPKI␣ in the Star-PAP complex implies that phosphoinositide-based signal transduction pathways may be able to modulate Star-PAP function.
Another PI-4,5-P 2 -sensitive protein found at nuclear speckles is the protein kinase CKI␣ (formerly known as casein kinase I␣) (17). The protein kinase activity of CKI␣ is specifically inhibited by PI-4,5-P 2 (18). CKI␣ is a member of the ubiquitously expressed CKI family of constitutively active Ser/Thrspecific protein kinases (19). CKI activity is modulated through the unique recognition motif (S/T)(P)XX(S/T), where S/T represents a phosphoserine or phosphothreonine in the Ϫ3 position (20). The activity of CKIs toward their substrates is regulated through this "priming" phosphorylation that precedes phosphorylation by CKIs. However, this is not the only mechanism of regulation for CKI family members.
Because the composition of a polyadenylation complex is important for the function of the associated poly(A) polymerase (5,9), we looked for additional unique components of the Star-PAP-associated complex relative to PAP␣. Identification of other unique Star-PAP-associated factors will lead to a more complete understanding of Star-PAP function and of its differences from other known PAPs. Here, we show that the Star-PAP complex contains unique protein kinase activities compared with PAP␣. One of these kinases is the PI-4,5-P 2sensitive kinase CKI␣, which is capable of directly phosphorylating Star-PAP. Moreover, CKI␣ and PIPKI␣ are required for the expression of a subset of Star-PAP target mRNAs. These data further reinforce the existence of a nuclear phosphoinositide-based signaling complex that regulates the Star-PAP polyadenylation complex to modulate expression of specific mRNAs.

EXPERIMENTAL PROCEDURES
Cell Culture, Transfection, and Treatments-HEK 293 and normal rat kidney cells were obtained from American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum at 37°C in 5% CO 2 . For DNA transfection, cells were transfected using the calcium phosphate method with the indicated amounts of DNA. The growth medium was exchanged after 4 h, and the cells were harvested at the indicated time. siRNA oligonucleotides were transfected using calcium phosphate at a final concentration of 120 nM oligonucleotide/ml of growth medium. Growth medium was replaced 6 h after transfection, and the transfection was repeated 24 h later. Cells were harvested for analysis 72 h after the first transfection. For PIPKI␣, the oligonucleotides used were PIPKI␣-1 (GGUGCCAUCCAGUUAGGCA) and PIPKI␣-3 (GAAGUUGGAGCACUCUUGG). For CKI␣, an siGENOME SMARTpool (Dharmacon) was directed against CSNK1A1. To induce a transcriptional antioxidant response, HEK 293 cells were treated with 100 M tert-butylhydroquinone (tBHQ; Sigma) in Me 2 SO for 4 h. Control cells were treated with Me 2 SO only. CKI inhibitors CKI-7 (Sigma) and IC261 (Calbiochem) were resuspended in Me 2 SO and used at the final concentrations indicated.
Immunofluorescence-Normal rat kidney cells were plated on glass coverslips in 35-mm dishes. Cells were transfected with 2.5 g of FLAG-Star-PAP and allowed to express for 24 h. Coverslips were washed in phosphate-buffered saline and fixed for 10 min in Ϫ70°C methanol. Immunofluorescence staining and microscopy were preformed as described previously (13) using anti-FLAG M2 (Sigma-Aldrich) and rabbit anti-CKI␣ polyclonal antibodies.
Expression and Affinity Purification of FLAG Proteins-Human Star-PAP and rat CKI␣ cDNAs were cloned into the pFLAG-1 mammalian expression vector (Sigma). The kinasedead CKI␣ mutant K46R was generated by PCR-based mutagenesis using primers 5Ј-gaagtggcagtgagactagaatcccag-3Ј and 5Ј-ctgggattctagtctcactgccactcc-3Ј. For each FLAG purification, ϳ5 ϫ 10 6 HEK 293 cells in four 10-cm dishes were transfected with 10 g of DNA and allowed to express for 48 h. FLAG purifications were preformed as described previously (6) according to the manufacturer's instructions.
Generation and Purification of Star-PAP Truncation Mutants-FLAG-tagged Star-PAP phosphorylation site point mutants were generated using PCR-based mutagenesis. The wild-type and mutant constructs were transfected into HEK 293 cells, allowed to express for 24 h, and lysed in FLAG lysis buffer (6). 2 g of anti-FLAG M2 antibody was added to the clarified lysates and incubated at 4°C with rotation for 1 h, followed by additional 1 h with protein A-Sepharose beads (Amersham Biosciences). The beads were washed three times with FLAG lysis buffer and resuspended in 1ϫ kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , and 0.5 mM EGTA) for use in kinase assays.
Protein Kinase Assays-Protein kinase assays were preformed in 1ϫ kinase buffer. Assays were initiated by the addition of 10 M ATP and 5 Ci of [␥-32 P]ATP to the reaction mixture. The endogenous kinase activity in the Star-PAP complex was destroyed by heating for 15 min at 65°C. For inhibitor studies, all reaction components except ATP were incubated with inhibitors for 45 min on ice prior to starting the assay. CKI inhibitors IC261 (IC 50 ϭ 11 M) and CKI-7 (IC 50 ϳ 6.0 M) were resuspended in Me 2 SO and used at final concentrations of 0.1-100 M. Synthetic PI-4,5-P 2 (Echelon Biosciences Inc.) was resuspended in 50 mM Tris-HCl (pH 7.9) at 2.5 mM, subjected to bath sonication to form micelles, and used at final concentrations of 12.5-100 M.
Quantitative Real-time PCR-This was preformed as described previously (6). All mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels.

RESULTS
The Star-PAP Complex Contains Protein Kinase Activity-Purification of FLAG-Star-PAP or FLAG-PAP␣ from HEK 293 cells resulted in the copurification of a large protein complex (6). This complex contained factors essential for the 3Ј-end formation of mRNAs, including CPSF 73 , CPSF 100 , CstF 64 , symplekin, and RNA polymerase II (6). In contrast to PAP␣, the Star-PAP complex also contained PIPKI␣ and, accordingly, PI-4-P kinase activity. To enhance the characterization of the Star-PAP complex, we subjected both the purified Star-PAP and PAP␣ complexes to an in vitro protein kinase assay to detect the presence of any associated protein kinases.
FLAG-Star-PAP and FLAG-PAP␣ were expressed in HEK 293 cells and purified on anti-FLAG M2 resin. Purified PAP complexes were subjected to an in vitro protein kinase assay with the generic protein kinase substrate myelin basic protein (MBP) or casein (100 g/ml). The purified Star-PAP complex contained protein kinase activity toward both MBP and casein, whereas the PAP␣ complex contained almost no detectable protein kinase activity (Fig. 1A). Interestingly, in these same assays, FLAG-Star-PAP was robustly phosphorylated (Fig. 1A), indicating that the associated kinase(s) can phosphorylate Star-PAP itself. These results identify protein kinase activity as another unique feature of the Star-PAP protein complex compared with PAP␣. Furthermore, the ability of this kinase activity to phosphorylate Star-PAP suggests that phosphorylation may play a role in Star-PAP function.
The Star-PAP complex includes PIPKI␣, and the activity of Star-PAP itself is directly regulated by the PIPKI␣ product PI-4,5-P 2 . Because of the relationship between Star-PAP and PI-4,5-P 2 , it is possible that other components of the Star-PAP complex may be regulated by PI-4,5-P 2 as well. Therefore, the sensitivity of Star-PAP phosphorylation by the associated kinase(s) to PI-4,5-P 2 was examined. Phosphorylation of FLAG-Star-PAP by the associated kinase(s) was inhibited by PI-4,5-P 2 at concentrations as low as 12.5 M (Fig. 1B), indicating that the associated kinase activity is indeed sensitive to PI-4,5-P 2 . Remarkably, this is in the same range of PI-4,5-P 2 concentrations that stimulate Star-PAP poly(A) polymerase activity (6), indicating that both protein kinase and Star-PAP activities are regulated by similar concentrations of PI-4,5-P 2 .
The PI-4,5-P 2 -sensitive Kinase CKI␣ Is Associated with the Star-PAP Complex-There are very few protein kinases known to be inhibited by PI-4,5-P 2 ; one of these is CKI␣ (17). Accordingly, immunoblotting of purified FLAG complexes with a CKI␣-specific antibody showed that CKI␣ copurified specifically with Star-PAP and not with PAP␣ ( Fig. 2A). In addition, immunoprecipitation of endogenous Star-PAP from HEK 293 cells resulted in coprecipitation of endogenous CKI␣ (Fig. 2B), demonstrating that these two proteins reside in the same complex in vivo. Both Star-PAP (6) and CKI␣ (17) have been reported to localize at nuclear speckles. Consequently, there was a strong colocalization between FLAG-Star-PAP and endogenous CKI␣ in the nuclei of normal rat kidney cells at nuclear speckles (Fig. 2C), demonstrating that in vivo Star-PAP and CKI␣ are present at the same sites within the nucleus and are therefore spatially positioned to interact with each other.
To confirm that the associated CKI␣ is involved in the phosphorylation of Star-PAP, the ability of CKI-specific inhibitors to block the phosphorylation of FLAG-purified Star-PAP by the associated kinase activity was examined. The CKI inhibitors IC261 (Fig. 2D) and CKI-7 (Fig. 2E) were both able to effectively block the phosphorylation of Star-PAP by the associated kinase activity in a dose-dependent fashion, signifying that CKI␣ is responsible for at least some of the kinase activity contained in the Star-PAP complex. The presence of CKI␣ specifically in the Star-PAP complex serves to further demonstrate that although Star-PAP and PAP␣ both contain the same basic 3Ј-end formation components, the Star-PAP complex contains additional signal transduction components that serve to distinguish it from the PAP␣ complex.
CKI␣ Can Directly Phosphorylate Star-PAP in Vitro on the Proline-rich Insert Region-The ability of IC261 and CKI-7 to block Star-PAP phosphorylation in the context of the purified complex suggests a role for CKI␣ in Star-PAP phosphorylation, but it does not prove that CKI␣ itself is directly phosphorylating Star-PAP. To demonstrate direct phosphorylation, purified CKI␣ was used to phosphorylate FLAG-purified Star-PAP. Prior to the assay, endogenous kinase activity in the FLAG complex was destroyed by heat inactivation. After heat inactivation, there was no detectable phosphorylation of Star-PAP (Fig. 3A). Purified FLAG-CKI␣ was able to directly phosphorylate heatinactivated Star-PAP, whereas the catalytically inactive CKI␣ mutant K46R (21) was not (Fig. 3A). Similarly, phosphorylation by CKI␣ was blocked by 50 M IC261 or 50 M PI-4,5-P 2 (Fig.  3B). Together, these data indicate that the phosphorylation of Star-PAP is occurring directly by CKI␣.
To determine the CKI␣ phosphorylation site(s) on Star-PAP, a series of FLAG-Star-PAP truncation and deletion mutants (Fig. 3C) was expressed and purified from HEK 293 cells and  MAY 2, 2008 • VOLUME 283 • NUMBER 18 subjected to the same in vitro kinase assays described above. CKI␣ was able to phosphorylate all truncation mutants except those that lacked the first half of the proline-rich region (⌬PRR 1/2, amino acids 223-274) that splits the catalytic domain of Star-PAP (Fig. 3D), demonstrating that this region contains the CKI␣ phosphorylation site(s) in Star-PAP. This region contains nine serine and threonine residues conserved among mammalian species, including two consensus CKI␣ sites and a number of acidic residues that could contribute to additional CKI␣ phosphorylation sites (Fig. 3E).

Phosphorylation of Star-PAP by CKI␣
The Star-PAP PRR Is Not Required for CKI␣ Association with the Star-PAP Complex-The above results leave open the possibility that CKI␣ does not phosphorylate Star-PAP in the PRR but, rather, that CKI␣ requires the PRR to associate with the Star-PAP complex and subsequently phosphorylates other sites within Star-PAP. To examine this, the requirement for the Star-PAP PRR for association of CKI␣ and for protein kinase activity with the Star-PAP complex was assessed. Both full-length Star-PAP and Star-PAP⌬PRR expressed and purified from HEK 293 cells associated with endogenous CKI␣ (Fig. 4A). Furthermore, although FLAG-purified Star-PAP⌬PRR could not be phosphorylated by the associated kinases (Fig. 4B), the complex still contained activity toward both casein and MBP similar to that of full-length Star-PAP (Fig. 4C), demonstrating that deletion of the PRR does not disrupt the association of protein kinase activity with the Star-PAP complex. This indicates that the inability of CKI␣ to phosphorylate Star-PAP⌬PRR mutants is most likely due to a deletion of the phosphorylation site(s) and not to a disruption of the Star-PAP/CKI␣ interaction.
CKI␣ and PIPKI␣ Are Required for the Expression of Specific Star-PAP Target mRNAs-Star-PAP and PIPKI␣ are required for expression of detoxifying and oxidative stress response mRNAs; however, microarray analysis indicated that Star-PAP is required for the expression of a diverse assortment of mRNAs (6). Therefore, the requirements for CKI␣ and PIPKI␣ for the expression of a diverse group of Star-PAP target mRNAs were analyzed. A group of mRNAs in which protein products function in a wide array of cellular processes was selected for further analysis, including heme oxygenase 1 (HO-1), NADPH:quinone oxidoreductase 1 (NQO1), cation transporter regulator-like 1 (CHAC1), asparagine synthetase (ASNS), p8 protein homolog (COM1), and secretogranin II (SCG2). The non-target mRNA glutamate-cysteine ligase, catalytic subunit (GCLC) was used as a negative control.
To confirm the requirement for Star-PAP in the expression of these mRNAs, HEK 293 cells were treated with siRNA oligonucleotides specific for Star-PAP. Knockdown of Star-PAP expression resulted in a 5-15-fold decrease in the mRNA species examined (Fig. 5, A and D), indicating that Star-PAP is indeed required for their expression. Treatment of HEK 293 cells with CKI␣-specific siRNA resulted in a dramatic decrease in HO-1 and NQO1 mRNAs, whereas other Star-PAP target mRNAs were unaffected (Fig. 5, B and  E). Likewise, we found that treatment of cells with PIPKI␣specific siRNA resulted in comparable decreases in the same Star-PAP target mRNAs as CKI␣ siRNA, viz. HO-1 and NQO1 (Fig. 5, C and F). Together, these data raise the possibility that PIPKI␣ and CKI␣ may be working together to regulate specific Star-PAP target mRNAs.
HO-1 and NQO1 mRNAs encode important detoxifying enzymes involved in protection from reactive oxygen species and cellular injury (22). Both HO-1 and NQO1 are up-regulated in response to oxidative stress through increased transcription (23). We have shown previously that Star-PAP and PIPKI␣ are required for the up-regulation of the HO-1 transcript in response to a tBHQ-induced antioxidant response by playing a direct role in the 3Ј-end formation of this mRNA (6). Pretreatment of cells with the CKI-specific inhibitors CKI-7 and IC261 not only reduced the basal levels of HO-1 mRNA but also effectively blocked HO-1 induction after exposure to 100 M tBHQ (Fig. 5E). Treatment of HEK 293 cells with CKI␣-specific siRNA oligonucleotides also caused a reduction in basal HO-1 levels; however, it did not block HO-1 induction by tBHQ (Fig. 5F). The requirement for CKI␣ and PIPKI␣ specifically for Star-PAP target mRNAs involved in cellular protection suggests that phosphoinositide-based signaling is involved in the Star-PAP regulation of stress response mRNA maturation. However, although CKI inhibitors can effectively block HO-1 induction, CKI␣-specific siRNA does not. This suggests that other CKI isoforms, or other protein kinases sensitive to CKI inhibitors, are also involved in the induction of HO-1 mRNA.
CKI␣ Associates Specifically with Star-PAP-dependent mRNAs-Although both Star-PAP and CKI␣ are required for the expression of the same mRNAs, it remains to be determined whether these proteins are actually acting together to regulate the levels of these mRNAs. Previously, we have shown that Star-PAP specifically interacts with its target mRNAs (6). Therefore, the ability of CKI␣ to interact with target mRNAs was examined by RNA immunoprecipitation (24).
Endogenous CKI␣ and Star-PAP were immunopurified from HEK 293 cells, and total RNA was isolated from the immunoprecipitates. Specific mRNAs were then detected using reverse transcription-PCR. Similarly to Star-PAP, CKI␣ was specifically associated with its putative target mRNA, HO-1 (Fig. 6).
Notably, CKI␣ did not interact with the Star-PAP target mRNA CHAC1, the expression of which does not require CKI␣ or PIPKI␣. This indicates that the association of CKI␣ with the Star-PAP complex occurs only with specific target mRNAs and that CKI␣ is not a universal component of all Star-PAP complexes.

DISCUSSION
Star-PAP is a novel poly(A) polymerase involved in the regulation of specific mRNA expression levels. Star-PAP differs from both the canonical and non-canonical poly(A) polymerases in several ways, including the presence of the PRR insert in its catalytic domain, a unique association with the PI-4,5-P 2generating enzyme PIPKI␣, and direct regulation by PI-4,5-P 2 .
Here, we show that Star-PAP also differs markedly from canonical PAP␣ because of the association of protein kinase activity with the purified enzyme complex and that the PI-4,5-P 2 -sensitive protein kinase CKI␣ is one of the kinases contributing to this activity. We also found that both PIPKI␣ and CKI␣ are required for the synthesis of the Star-PAP target mRNAs HO-1 and NQO1, which are vital protective enzymes that respond to reactive oxygen species and cellular oxidative stress (22). Furthermore, like Star-PAP, CKI␣ is associated specifically with these mRNAs, suggesting that Star-PAP and CKI␣ work together to regulate their expression.
Phosphoinositide signaling is based on the subcellular targeting of phosphoinositide-generating enzymes and the subsequent production of phosphoinositide signaling molecules at specific subcellular sites (12). The presence of multiple PI-4,5-P 2 -sensitive components, including Star-PAP and CKI␣, as well as the PI-4,5-P 2 -generating enzyme PIPKI␣, suggests that the Star-PAP complex is a focal point for phosphoinositide signaling in the nucleus. Localized PI-4,5-P 2 generation by PIPKI␣ is likely able to modulate the activities of both CKI␣ and Star-PAP itself, which could result in the regulation of Star-PAP function by phosphoinositides at multiple levels.
It is interesting to note that the Star-PAP complex contains a number of proteins that are phosphorylated in this assay, none of which are present in the PAP␣ complex (Fig. 1A). As with Star-PAP, phosphorylation of these proteins is also inhibited by PI-4,5-P 2 . This suggests that additional components of the Star-PAP complex may also be regulated by PI-4,5-P 2 -sensitive protein phosphorylation.
Combined, these data point to the Star-PAP complex as a site where multiple PI-4,5-P 2 -sensitive components are present to take advantage of locally generated PI-4,5-P 2 to regulate Star-PAP function. The presence of phosphoinositides in the nucleus has been well established; however, until recently, the mechanisms by which they affected nuclear events were unknown (25). The data presented here help to establish the mechanism by which nuclear phosphoinositides generated at nuclear speckles regulate mRNA processing and expression.
CKI␣ and PIPKI␣ are required for the expression of some but not all Star-PAP target mRNAs. This suggests that phosphoinositide-based signal transduction does not regulate all aspects of Star-PAP function and, additionally, that maturation of all Star-PAP target mRNAs is not regulated through the same signaling pathway. Furthermore, the fact that the related Star-PAP target mRNAs HO-1 and NQO1 require CKI␣ and PIPKI␣, whereas unrelated Star-PAP target mRNAs such as CHAC1 do not, reveals that maturation of Star-PAP target mRNAs may be divided into groups based on their function and regulation. Star-PAP target mRNAs may exist as distinct groups, or modules, each of which is regulated by a different signal transduction pathway. For example, the HO-1/NQO1 oxidative stress response module is regulated by a CKI␣and PIPKI␣-based phosphoinositide pathway, whereas other modules may be controlled by different pathways. This arrangement would allow Star-PAP to specifically affect the expression of individual mRNA groups.
It is also notable that CKI␣ is associated only with Star-PAP target mRNAs, the levels of which it affects. This indicates that the composition of the Star-PAP 3Ј-processing complex is dynamic and can change to regulate different groups of mRNAs. It is therefore likely that other, as yet unidentified signal transduction components can also be associated in a Star-PAP complex. This suggests a model in which different signal transduction components assemble into the Star-PAP complex to control the expression of specific Star-PAP target mRNA modules. Each of these different Star-PAP complexes would be targeted to a specific Star-PAP module. The activation of specific signal transduction pathways could then direct Star-PAP to participate in the 3Ј-end formation of select mRNAs to which that specific complex was targeted while not affecting the expression of the others. In this manner, Star-PAP would be positioned to interpret an array of stimuli and coordinate the cellular response by regulating mRNA expression.
It is still unclear how the Star-PAP complex identifies different mRNA modules. It could be that incorporation of certain factors directs the complex to different mRNA targets. Alternatively, association of the Star-PAP complex with members of each mRNA group could recruit different sets of signal transduction components. Identification of more Star-PAP target mRNAs and their inherent modules will be necessary before the mechanism of Star-PAP specificity can be explored.
Although basal expression of HO-1 requires both PIPKI␣ and CKI␣, CKI␣ is not required for HO-1 induction, suggesting overlapping but unique roles for each protein in Star-PAP regulation. However, CKI inhibitors are effective in blocking tBHQ-induced HO-1 mRNA expression. This raises the possibility that other CKI isoforms can compensate for the loss of CKI␣ and can allow Star-PAP-dependent induction of HO-1 mRNA expression. Alternatively, it may be that other CKI isoforms, or even other CKI inhibitor-sensitive protein kinases, are a functional component of the Star-PAP complex and play a role in Star-PAP-dependent HO-1 mRNA induction.
The location of the CKI␣ phosphorylation site in the PRR of Star-PAP that interrupts the catalytic domain means that phosphorylation of Star-PAP could affect its catalytic activity. This could be through either a direct effect of the phosphorylation on activity or the modulation of an interaction with yet unknown regulatory proteins. Alternatively, it could be that this site is somehow important for substrate recognition or even possibly involved in PI-4,5-P 2 binding and modulation of Star-PAP activity. Deletion of either the entire PRR or the portion that contains the CKI␣ phosphorylation site(s) results in a complete loss of Star-PAP poly(A) polymerase activity (data not shown). This may be due to a loss of regulatory elements, but just as likely, it may be due to a general disruption of the structure of the catalytic core of the Star-PAP enzyme.
Phosphorylation of PAP␣ has been shown to modulate its activity (26). However, on PAP␣, this phosphorylation occurs on a serine/threonine-rich region in its carboxyl terminus. A comparable region does not exist in the same location on Star-PAP, so it is likely that the mechanism by which phosphorylation affects Star-PAP function will be different from that by which phosphorylation affects the activity of PAP␣. We have not been able to identify the specific CKI␣ phosphorylation site on Star-PAP using individual point mutants. It is possible that CKI␣ is phosphorylating Star-PAP on multiple sites within the PRR. To gain a better understanding of how CKI␣ phosphorylation affects Star-PAP function, these site(s) must be identified.
Although CKI␣ is one protein kinase component of the Star-PAP complex, it is likely not the only one. The Star-PAP complex contains activity toward both MBP and casein. However, CKI␣ is unable to directly phosphorylate MBP, suggesting that there is at least one other protein kinase associated with the Star-PAP complex. Additionally, CKI inhibitors are capable of inhibiting casein phosphorylation by the associated kinase activity but are ineffective in blocking MPB phosphorylation (data not shown). Finally, most CKI␣ substrates must first be phosphorylated by another kinase to generate a CKI␣ phosphorylation site (20). Notably, CKI␣ is unable to phosphorylate Escherichia coli-expressed, purified Star-PAP (data not shown), which would not be expected to contain the priming phosphorylation.
Together, these data strongly suggest that another kinase is also associated with the purified Star-PAP complex. However, the contribution of this kinase to Star-PAP phosphorylation is still unclear. CKI inhibitors are able to block a majority of Star-PAP phosphorylation in vitro; however, the ability of this kinase to phosphorylate Star-PAP in vivo and/or afterward in response to specific stimuli cannot be discounted. It may be that this unidentified kinase activity represents the CKI␣ priming kinase. Alternatively, this kinase may be involved in the regulation of other Star-PAP mRNAs, consistent with our model in which distinct Star-PAP complexes are each responsible for controlling the expression of specific mRNA modules.
The data presented here demonstrate an unexpected new nuclear substrate and function for the PI-4,5-P 2 -sensitive protein kinase CKI␣. CKI␣ can directly phosphorylate Star-PAP and is required for the expression of the Star-PAP target mRNAs HO-1 and NQO1, which encode critical detoxifying enzymes. The involvement of Star-PAP, PIPKI␣, and CKI␣ in the expression of these mRNAs suggests that a nuclear phosphoinositide signaling pathway regulates the level of specific mRNAs in response to oxidative stresses. However, the finding that phosphoinositide-based regulation of Star-PAP affects only a subset of Star-PAP target mRNAs demonstrates that Star-PAP is a very sophisticated regulatory enzyme. The association of the basic Star-PAP 3Ј-processing complex with distinct signal transduction components allows Star-PAP to specifically regulate the expression of discrete groups of mRNAs. In this manner, as depicted in Fig. 7, Star-PAP is capable of interpreting signals from distinct pathways and translating them into unique mRNA expression profiles by modulating the 3Ј-end formation of specific groups of Star-PAP target mRNAs.