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Originally published In Press as doi:10.1074/jbc.M413263200 on February 22, 2005

J. Biol. Chem., Vol. 280, Issue 16, 16522-16527, April 22, 2005
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A Central Kinase Domain of Type I Phosphatidylinositol Phosphate Kinases Is Sufficient to Prime Exocytosis

ISOFORM SPECIFICITY AND ITS UNDERLYING MECHANISM*

Li Wang, Gang Li, and Shuzo Sugita{ddagger}

From the Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network and Department of Physiology, University of Toronto, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada

Received for publication, November 24, 2004 , and in revised form, February 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exocytosis, a critical process for neuronal communication and hormonal regulation, involves several distinct steps including MgATP-dependent priming (which involves the synthesis of phosphatidylinositol 4,5-bisphosphate). Type I phosphatidylinositol phosphate kinases (PIPKIs) were purified biochemically as a priming factor. PIPKI consists of three domains: the N-terminal region, the central kinase domain, and the C-terminal region. Three isoforms ({alpha}, {beta}, and {gamma}) of PIPKI have been identified, and each is alternatively spliced at the C-terminal region. In the present study, we conducted a structure/function analysis of PIPKIs in the priming of exocytosis, and we found that recombinant PIPKI{alpha} and PIPKI{gamma} had priming activity. However, an unexpected finding of these results was that PIPKI{beta} did not prime exocytosis. The N- or C-terminal region of PIPKI{alpha} and PIPKI{gamma} was not required for priming, which indicates that the central kinase domain is sufficient for this process. Alternative splicing in each isoform did not affect the isoform specificity in priming. Priming activity by isoforms is strongly correlated with their phosphatidylinositol phosphate kinase activity because PIPKI{alpha} and PIPKI{gamma} had higher kinase activity than PIPKI{beta}. These results suggest that PIPKI{alpha} and PIPKI{gamma} are the critical priming factors for exocytosis; it also suggests that the levels of phosphatidylinositol phosphate kinase activity in producing phosphatidylinositol 4,5-bisphosphate specify the function of PIPKI isoforms in priming.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurotransmitter exocytosis is regulated by many cytosolic and membrane proteins (1–3). A reconstituted exocytosis assay using permeabilized PC12 cells has proved very effective in the molecular dissection of exocytosis (4–14). This assay revealed two kinetically distinct processes: MgATP-dependent priming and Ca2+-dependent triggering (4). Purification of priming factors identified PIPKIs1 and phosphatidylinositol transfer protein (6, 7). Ca2+-dependent activator protein for secretion was identified as a triggering factor (5). The recognition of a role for PIPKI and phosphatidylinositol transfer protein has led researchers to hypothesize that PIP2 generation by PIPKI and phosphatidylinositol transfer protein, along with membrane-associated phosphatidylinositol 4-kinase (15), is a key component of exocytosis.

Molecular cloning has identified three isoforms of PIPKI: {alpha}, {beta}, and {gamma} (16–18). Two different research groups, working independently, have simultaneously given the same isoforms different names ("PIPKI{alpha}" and "PIPKI{beta}") (16, 17). In this article, we will follow the nomenclature of Loijens and Anderson (17). PIPKI{gamma} is expressed primarily in the brain (18), where it is concentrated at the synapse of the neurons (19); PIPKI{alpha} and PIPKI{beta}, by contrast, are expressed ubiquitously (16, 17). The in vivo function of PIPKI{gamma} has been investigated recently through the generation of PIPKI{gamma} knock-out mice. The mice die after birth, and their vesicle trafficking at the synapse is severely disrupted (20).

Each PIPKI isoform is alternatively spliced at the C-terminal region (Refs. 16 and 17 and the present study). The splicing sequence (residues 636–661) in PIPKI{gamma} is critical for binding to talin (21, 22), which is a component of focal adhesion plaques. It was found that only the longer isoform of PIPKI{gamma} concentrates at focal adhesion points of the cell via a talin link, where it enhances the PIP kinase activity of PIPKI{gamma}. What has not been examined, however, is whether this alternative splicing also regulates the priming activity of PIPKI.

Overexpression of PIPKI isoforms in COS7 cells results in massive actin polymerization (16, 23); this suggests that PIP-KIs may also be involved in the regulation of actin cytoskeleton, in addition to their role in membrane trafficking. Overexpression of PIPKI{alpha}, but not of PIPKI{beta}, has effects on the endocytosis of the epidermal growth factor receptor (24). Similar isoform specificity may exist in PIPKI-mediated priming: Aikawa and Martin (25) found that the transfection of PC12 cells with PIPKI{alpha} or PIPKI{gamma}, but not with PIPKI{beta}, reversed the ARF6 transfection-mediated inhibition of priming. However, the authors of that study could not draw a conclusion about this isoform specificity, given that they found that the expression level of transfected PIPKI{beta} in PC12 cells was lower than it was in other isoforms (25). It is unclear what determines these potential isoform specificities. In this article, we show that bacterially expressed recombinant PIPKIs exhibit priming activity. Using these recombinant PIPKIs, we have addressed the isoform specificity as well as the mechanism that underlies it. We also attempted to determine whether alternative splicing in PIPKIs affects their priming activity, and whether the central kinase domain of PIPKIs is sufficient for the priming of exocytosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
PC12 Secretion Assay—PC12 cells were maintained in 10-cm dishes with 8 ml of Dulbecco's modified Eagle's medium containing 5% calf serum (Hyclone), 5% horse serum (Hyclone), and 100 units/ml penicillin and streptomycin (Sigma) at 37 °C in 9.5% CO2. The secretion assay followed the protocols of previously published work (4). PC12 cells were labeled for 12–20 h with 4 µl of [3H]norepinephrine (NE; 56.4 Ci/mmol; PerkinElmer Life Sciences) in the presence of 0.5 mM ascorbic acid. After washing, the cells were harvested in KGlu buffer (20 mM HEPES, pH 7.2, 120 mM potassium glutamate, 20 mM potassium acetate, and 2 mM EGTA) with 0.1% bovine serum albumin, permeabilized with a ball homogenizer (H&Y Enterprise), and incubated for 1–3 h on ice, in the presence of 10 mM EGTA, in order to extract the cytosolic proteins. Two-stage secretion assays were performed in KGlu buffer with 0.05–0.1% bovine serum albumin. Thirty-minute priming incubations at 30 °C contained permeabilized PC12 cells, 2 mM MgATP, and recombinant proteins (or 1.0 mg/ml rat brain cytosol). The cells were recovered by centrifugation, washed with KGlu buffer with 0.1% bovine serum albumin, and used for 5-min triggering incubations at 30 °C that contained Ca2+ (1.72 mM; free Ca2+ concentrations are estimated to be ~1–10 µM) and 0.5 mg/ml rat brain cytosol, which provides the Ca2+-dependent activator protein for secretion required for triggering (5).

Construction of Expression Plasmids—All the expression plasmids were generated using parental plasmid pGex-KG (9, 14). Expression plasmids for full-length PIPKIs and PIPKII{alpha} are pGex-mPIPKI{alpha}-1 (a long form of PIPKI{alpha}, made from IMAGE clone 4503697), pGex-mPIPKI{alpha}-8 (a short form of PIPKI{alpha} generated by PCR on pGex-mPIPKI{alpha}-1 based on the sequence information from IMAGE clones 30932032 and 30608358), pGex-mPIPKI{beta}-3 (a short form of PIPKI{beta}, made from IMAGE clone 3326897), pGex PIPKI{beta}-4 (a long form of PIPKI{beta}, made from IMAGE clone 5289812), pGex-mPIPKI{gamma}-3 (a short form of PIPKI{gamma}, made from IMAGE clone 4459567), pGex-hPIPKI{gamma}-5 (a long form of PIPKI{gamma}, made from clone KIAA 0589), and pGex-mPIP-KII{alpha}-1 (PIPKII{alpha}, made from IMAGE clone 3672732). IMAGE clone 4459567 contained a mutation at residue 12 (S12A), which was corrected by a mutagenesis kit (Stratagene). IMAGE clones were purchased from Invitrogen. KIAA 0589 was a kind gift from Dr. Takahiro Nagase (Kazusa DNA Research Institute). Expression constructs for truncated PIPKIs include pGex-mPIPKI{alpha}-3 (encoding residues 1–439 of PIPKI{alpha}, C-terminal truncation), pGex-mPIPKI{alpha}-4 (residues 32–439 of PIPKI{alpha}, N- and C-terminal truncation), and pGex-mPIPKI{gamma}-2 (residues of 52–635 of PIPKI{gamma}, N-terminal truncation).

Expression and Purification of GST Fusion Proteins—Proteins were expressed in Origami B (DE3) strain (Novagen) or Origami B (DE3) harboring pG-Tf2 (Takara) and purified with glutathione-agarose (Sigma) (9, 14), which in some cases included MgATP washing. Proteins were eluted with an elution buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, and 10 mM glutathione), and the eluted proteins were then concentrated by centrifugation with Nanosep 30 (Pall Gelman). Concentrated proteins (~0.2 mg/ml) were dialyzed with KGlu buffer. In some cases, GST fusion proteins were cleaved by thrombin (Roche Applied Science) in a cleavage buffer (50 mM Tris, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2). Cleaved proteins were then processed for the secretion assay, similar to the eluted proteins.

Kinase Activity Assays—PIP kinase activity was measured in 50-µl reactions, performed for 11 min at room temperature in a final concentration of 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 80 µM phosphatidylinositol 4-phosphate (Sigma), 50 µM ATP, and 10 µCi of [{gamma}-32P]ATP. The amounts of recombinant proteins used were 0.4 µg for GST and GST-PIPKIs. The reaction was stopped by the addition of 100 µl of 1 N HCl, and lipids were then extracted with 200 µl of chloroform/methanol (1:1). This was followed by further extraction with 80 µl of methanol/1 N HCl (1:1). The extracted lipids were separated using thin layer chromatography plates (Silica Gel 60; Merck) in chroloform/methanol/15 N NH4OH/distilled H2O (90:90:7:22), and the labeled products were detected by autoradiography (16, 17).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Recombinant PIPKI{alpha} Primes Exocytosis—First, we replicated the two-stage assay using permeabilized PC12 cells (4) (Fig. 1). As previously described, incubation of the PC12 cell ghosts with brain cytosol and MgATP at the priming stage enhanced Ca2+-dependent neurotransmitter exocytosis at the triggering stage. Omission of either brain cytosol or MgATP during the priming stage resulted in poor Ca2+-dependent exocytosis during the triggering stage (Fig. 1). Incubation with brain cytosol plus MgATP at the priming stage resulted in an ~3-fold increase in exocytosis at triggering, compared with incubation with MgATP alone at the same stage (Fig. 1) (t10 = 11.3, p < 0.0001). This significant effect provided sufficient resolution to allow us to examine the effect of an individual priming factor.



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  		FIG. 1.
Establishment of two-stage assay using permeabilized PC12 cells. Both MgATP and brain cytosol are required for efficient priming for NE release. The permeabilized PC12 cell ghosts were primed by incubation for 30 min with MgATP ({square}), MgATP plus brain cytosol ({blacksquare}), or brain cytosol alone (). The primed cells were washed once and incubated for 5 min with brain cytosol ± Ca2+ at the triggering stage. Error bars indicate S.E. (n = 6).

 
We expressed full-length mouse PIPKI{alpha} as a GST fusion protein (GST-mPIPKI{alpha}-1) in Escherichia coli. The purified protein was eluted with glutathione and dialyzed with the same buffer (KGlu buffer) used in the secretion assay (see "Experimental Procedures") (Fig. 2A). We questioned whether the recombinant PIPKI{alpha} alone (i.e. without brain cytosol) could significantly prime exocytosis. Purified GST-mPIPKI{alpha}-1 and MgATP were introduced to permeabilized PC12 cells at the priming stage, and their effects on exocytosis at the triggering stage were examined. Recombinant PIPKI{alpha} exhibited dose-dependent priming activity and could prime up to 60% above the control (Fig. 2B), whereas purified GST had no effect. The dose required for priming was similar to the required dose of partially purified native PIPKI from erythrocytes (7), suggesting that recombinant PIPKI{alpha} and native PIPKI are similarly active in priming. We were able to conclude that PIPKI{alpha} is indeed an important factor for priming and that recombinant PIPKIs are useful tools for the study of the structure/function relationship of PIPKI in priming of neurotransmitter exocytosis.



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  		FIG. 2.
Recombinant PIPKI{alpha} primes neurotransmitter exocytosis. A, analysis of purified GST (~2 µg) and full-length mouse PIPKI{alpha} fused with GST (GST-mPIPKI{alpha}-1) by SDS-PAGE and Coomassie Blue staining. In this gel, the amount of full-length product of GST-PIPKI{alpha}-1 was estimated to be ~2 µg. B, GST-PIPKI{alpha}-1 (but not GST) primed NE release. At the priming stage, PC12 cells were incubated with MgATP and the indicated amounts of either GST or GST-mPIPKI{alpha}-1. The primed cells were washed and incubated with Ca2+ and brain cytosol to trigger NE release. As a result of incubation with only MgATP during priming, the average NE release was set at 100% for each experiment. Error bars indicate S.E. (n = 4).

 
Isoform Specificity in Priming of Exocytosis and the Role of Alternative Splicing—Molecular cloning revealed three isoforms of PIPKIs, and each isoform is alternatively spliced. In PIPKI{gamma}, the C terminus is alternatively spliced (shown as D in Fig. 3A) (18), and this splicing regulates binding to the focal adhesion protein, talin (21, 22). This binding in turn augments the PIP kinase activity of PIPKI{gamma}. The C terminus of PIPKI{beta} is similarly spliced (shown as C in Fig. 3A) (17). In contrast, human PIPKI{alpha} is alternatively spliced at the region in the immediate proximity of the central kinase domain (shown as A in Fig. 3A). We found a nearly identical splicing in mouse PIPKI{alpha} by searching the mouse expressed sequence tag data base (Fig. 3B). In addition, we found a novel splicing site in mouse PIPKI{beta} (shown as B in Fig. 3A). We compared splicing site A in (human and mouse) PIPKI{alpha} and splicing site B in PIPKI{beta} by aligning the sequences of PIPKI{alpha} and PIPKI{beta}; we found that these two splicing sites exist at similar positions (Fig. 3B). Thus, the region in the immediate proximity of the central kinase domains of both PIPKI{alpha} and PIPKI{beta} is alternatively spliced by a similar mechanism; this finding suggests that there is a functional significance.



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  		FIG. 3.
Analysis of alternative splicing in C-terminal regions of PIPKIs. A, schematic representation of PIPKI isoforms. The sites of alternative splicing (labeled A–D) in C-terminal regions of PIPKIs are underlined. B, sequence alignment of splicing site A in PIPKI{alpha} and splicing site B in PIPKI{beta}. Sequences of human and mouse PIPKI{alpha} and PIPKI{beta} are aligned (h, human; m, mouse). Residues that are identical in at least three sequences are highlighted. The number of the last residue of each PIPKI sequence is indicated on the right. Alternative splicing site A found in human and mouse PIPKI{alpha} (Ref. 17 and this study) and splicing site B found in mouse PIPKI{beta} in this study are underlined.

 
To examine isoform specificity in the priming of exocytosis and investigate the role of alternative splicing for this process, we generated a short (i.e. lacking splicing site A) form of PIPKI{alpha}, long (i.e. containing splicing site B) and short (i.e. lacking splicing site B) forms of PIPKI{beta}, and long (i.e. containing splicing site D) and short (i.e. lacking splicing site D) forms of PIPKI{gamma} (Fig. 4, A and B). As a negative control, we generated recombinant PIPKII{alpha} (Fig. 4, A and B) because native PIPKII was previously shown to exhibit no priming activity (7). As expected, recombinant PIPKII{alpha} (GST-mPIPKII{alpha}-1) had no priming activity (Fig. 4C). In contrast, the short form of recombinant PIPKI{alpha} (GST-mPIPKI{alpha}-8) exhibited significant priming, as did the long form of PIPKI{alpha} (GST-mPIPKI{alpha}-1) (Fig. 4C). Furthermore, both the short (GST-mPIPKI{gamma}-3) and long (GST-hPIPKI{gamma}-5) forms of PIPKI{gamma} exhibited similar priming. Unexpectedly, neither the short (GST-mPIPKI{beta}-3) nor long (GST-mPIPKI{beta}-4) form of PIPKI{beta} exhibited priming activity (Fig. 4C). Thus, alternative splicing in each isoform does not appear to affect the priming of exocytosis. Furthermore, PIPKI{alpha} and PIPKI{gamma}, but not PIPKI{beta}, appear to be the priming factors for exocytosis.



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  		FIG. 4.
PIPKI{alpha} and PIPKI{gamma}, but not PIPKI{beta} or PIPKII{alpha}, prime exocytosis. A, schematic representation of full-length GST fusion PIPKI and PIPKII{alpha} proteins that are expressed and tested for priming of exocytosis. B, analysis of purified GST fusion PIPKI{alpha}, PIPKI{beta}, PIPKI{gamma}, and PIPKII{alpha} proteins by SDS-PAGE and Coomassie Blue staining. C, PIPKI{alpha} and PIPKI{gamma} exhibited priming activity, whereas PIPKI{beta} and PIPKII{alpha} did not. For each experiment, 1–2 µg of GST or GST-PIPKIs or 4 µg of GST-PIPKII was used to prime PC12 cells in the presence of 2 mM MgATP. The data were normalized so that the average of NE release as a result of priming by GST + MgATP (control; denoted by C in the figure) was set to 100% for each experiment. Error bars indicate S.E. (n = 9–21).

 
PIPKI{beta} has a shorter N-terminal sequence than PIPKI{alpha} and PIPKI{gamma} (Fig. 3A), raising the possibility that the N-terminal-linked GST might hinder PIPKI{beta} action. We cleaved GST from both GST-mPIPKI{beta}-4 and GST-mPIPKI{alpha}-1 using thrombin, and then we tested the cleaved PIPK{alpha} and PIPKI{beta} in priming (Fig. 5). Isoform specificity remained because PIPKI{alpha}, but not PIPKI{beta}, was still able to exhibit priming. From this, we concluded that isoform specificity is inherent to PIPKIs and is not solely due to a GST artifact.



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  		FIG. 5.
Isoform specificity remains after the cleavage of GST. GST-cleaved PIPKI{alpha}, but not GST-cleaved PIPKI{beta}, primed NE exocytosis. Error bars indicate S.E. (n = 8–10).

 
A Central Kinase Domain Is Sufficient to Prime Exocytosis—We also attempted to determine the critical domains of PIPKIs for priming. Previous deletion analysis of PIPKIs has determined the minimal kinase domains of PIPKIs (18), and all PIPKI isoform kinase domains are located in the central region (Fig. 3A). PIPKIs contain N-terminal and C-terminal regions that may be critical for protein-protein interactions (21, 22), as well as other functions. We suspected that the N-terminal region might contribute to the observed isoform specificity because, as noted above, PIPKI{alpha} and PIPKI{gamma} have a longer N-terminal region than PIPKI{beta} does. To test this possibility, we generated the N-terminal truncation protein of PIPKI{gamma} (Fig. 6A). The truncated PIPKI{gamma} (GST-mPIPKI{gamma}-2) exhibited robust priming that was comparable to the priming of the full-length PIPKI{gamma} (Fig. 6B). We also generated a C-terminal truncation for PIPKI{alpha} (Fig. 6A), and we were able to identify strong priming by the truncated PIPKI{alpha} (GST-mPIPKI{alpha}-3, Fig. 6C). Furthermore, the truncation of both N- and C-terminal regions of PIPKI{alpha} (GST-PIPKI{alpha}-4) did not reduce priming, indicating that the kinase domain of PIPKI{alpha}, and perhaps PIPKI{gamma} as well, is sufficient for priming (Fig. 6C).



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  		FIG. 6.
Central kinase domain of PIPKI{alpha} or PIPKI{gamma} is sufficient for priming. A, the schematic representation of truncated proteins of PIPKI{alpha} and PIPKI{gamma}. B and C, for comparison, the truncated GST-PIPKI{alpha} and GST-PIPKI{gamma} proteins were tested with GST (negative control) and GST full-length PIPKI{alpha} or PIPKI{gamma} (positive control). Error bars indicate S.E. (n = 6–9).

 
Isoform Specificity Is Determined by PIP Kinase Activity— Our finding of unexpected isoform specificity in priming raised two possibilities. First, the generation of PIP2 alone may be an inadequate explanation for the priming actions of PIPKIs (assuming that they all have comparable PIP kinase activity). The second possibility is that differences in the degree of kinase activity among PIPKI isoforms may underlie the differences in priming. To further examine these two possibilities, we compared the kinase activity of the recombinant PIPKI isoforms using phosphatidylinositol 4-phosphate as a substrate. Thin layer chromatography was used to separate the PIPKI phospholipid products. We found dramatic differences in PIP kinase activity among isoforms of PIPKI (Fig. 7). Both GST-mPIPKI{alpha}-1 and GST-mPIPKI{gamma}-3 exhibited strong kinase activity. In contrast, GST-mPIPKI{beta}-4 showed much lower kinase activity. Control experiments using GST alone did not show any kinase activity. These results indicate a strong correlation between PIP kinase activity and the priming of exocytosis; this suggests that the lack of priming activity by PIPKI{beta} is due to its relatively poor kinase activity. Our results also support the hypothesis that PIP2 generation is the critical element for priming.



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  		FIG. 7.
PIP kinase activity of PIPKI{beta} is dramatically lower than that of PIPKI{alpha} and PIPKI{gamma}. Autoradiography of thin layer chromatography from the PIP kinase assay demonstrates the differences in PIP kinase activity among PIPKI isoforms. Each recombinant protein tested was 0.4 µg, in a 50-µl total reaction. GST was used as a negative control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Two PIPKIs of 68 and 90 kDa were partially purified as priming factors for neurotransmitter exocytosis in permeabilized PC12 cells, with the 90-kDa protein having the major activity (7). Molecular cloning of PIPKI (16–18) followed this finding; it is worth recalling that priming by recombinant PIPKI has never been demonstrated. In addition, a structure/function analysis of PIPKIs has been unavailable. The present study has succeeded in demonstrating the priming activity of recombinant PIPKI{alpha} and PIPKI{gamma}. Our results suggest that the partially purified 68-kDa protein corresponds to PIPKI{alpha}, whereas the 90-kDa protein corresponds to PIPKI{gamma}. The results also imply that the predominant activity of the 90-kDa PIPKI, in the course of purification from brain cytosol (7), is due to the fact that the presence of PIPKI{gamma} in the brain is greater than that of PIPKI{alpha}, rather than the higher priming activity of PIPKI{gamma}. Furthermore, we have shown that the kinase domain of PIPKI{alpha} (and probably PIPKI{gamma}) alone is sufficient for priming. Unexpectedly, we found that PIPKI{beta} had little or no priming activity, compared with PIPKI{alpha}. This result could be attributed to the lower PIP kinase activity of PIPKI{beta}, compared with PIPKI{alpha} and PIPKI{gamma} (Fig. 7).

Similar isoform specificity has been suggested in other functions of PIPKIs. In particular, the effects of PIPKI{alpha} overexpression on the endocytosis of epidermal growth factor receptor may be another such function (24). Because epidermal growth factor receptor endocytosis requires the kinase activity of PIPKI{alpha}, the lack of an effect upon the overexpression of PIPKI{beta} may be due to its lower kinase activity. In contrast, all the isoforms of PIPKI were able to induce similar levels of actin polymerization upon overexpression (18, 23). Unexpectedly, actin polymerization by PIPKI seems to be independent of kinase activity (18, 23). Thus, the function of PIPKI isoforms may be differentially regulated (depending on the requirements of their PIP kinase activity), although the molecular mechanisms defining the isoform specificity in PIP kinase activity require further study.

It has been found that the C-terminal regions of PIPKI isoforms are alternatively spliced. We found a novel splicing site just in the immediate proximity of the central kinase domain in PIPKI{beta}, which is nearly identical to the site in PIPKI{alpha} (Fig. 3). We tested the function of these splicing sites in PIPKI{alpha} and PIPKI{beta}, as well as splicing in the C terminus of PIPKI{gamma}, in the regulation of their priming activities. However, our permeabilized secretion assays did not reveal any such regulation by alternative splicing. This result supports our finding that the central kinase domain alone is sufficient to prime exocytosis. But this does not exclude the possibility that the splicing may be critical for secretion from intact cells.

Our results support the hypothesis that PIP2 generation is the critical step in the priming of exocytosis. The generated PIP2 may serve as the signal for neurotransmitter release by interacting with the potential Ca2+ sensors for exocytosis (such as synaptotagmin (9–12, 26) and Ca2+-activating proteins (5, 27)). The localization of generation of PIP2, however, remains to be determined. The original hypothesis was that PIP2 is generated at the vesicles where major phosphatidylinositol phosphate 4-kinase activity is observed (15). However, recent experiments using the GFP-PH (pleckstrin homology) domain, which specifically binds to PIP2, suggest that PIP2 is located mainly at the plasma membrane (25, 28). The hypothesis that PIP2 is generated at the plasma membrane is supported by the revelation that transfected PIPKI attaches to the plasma membrane (29). Our active recombinant PIPKIs will be a useful tool in future investigations of the localization of PIP2 generation in permeabilized PC12 cells.


   FOOTNOTES
 
* This work was supported by Canada Research Chair program and Canadian Institute of Health Research Grant MOP57825 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Toronto Western Research Institute, MC11-432, University Health Network, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada. Tel.: 416-603-5077; Fax: 416-603-5745; E-mail: ssugita{at}uhnres.utoronto.ca.

1 The abbreviations used are: PIPKI, type I phosphatidylinositol phosphate kinase; PIPKII, type II phosphatidylinositol phosphate kinase; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; NE, [3H]norepinephrine; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Erik Schweitzer (University of California Los Angeles) and Thomas Martin (University of Wisconsin) for kindly providing us with the PC12 cell line, which was originally isolated by Dr. Schweitzer. We are also grateful to Dr. Takahiro Nagase for allowing us to use the KIAA 0589 clone. We also thank Lakshmanan Arunachalam and Drs. James Eubanks and Elise Stanley (Toronto Western Research Institute) for critical reading of an earlier version of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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