Decoding of short-lived Ca2+ influx signals into long term substrate phosphorylation through activation of two distinct classes of protein kinase C.

In electrically excitable cells, membrane depolarization opens voltage-dependent Ca(2+) channels eliciting Ca(2+) influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca(2+) influx alone can activate PKC. The present study was conducted to investigate the Ca(2+) influx-induced activation mechanisms for two classes of PKC, conventional PKC (cPKC; PKCalpha) and novel PKC (nPKC; PKCtheta), in insulin-secreting cells. We have demonstrated simultaneous translocation of both DsRed-tagged PKCalpha to the plasma membrane and green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate to the cytosol as a dual marker of PKC activity in response to depolarization-evoked Ca(2+) influx in the DsRed-tagged PKCalpha and GFP-tagged myristoylated alanine-rich C kinase substrate co-expressing cells. The result indicates that Ca(2+) influx can generate diacylglycerol (DAG), because cPKC is activated by Ca(2+) and DAG. We showed this in three different ways by demonstrating: 1) Ca(2+) influx-induced translocation of GFP-tagged C1 domain of PKCgamma, 2) Ca(2+) influx-induced translocation of GFP-tagged pleckstrin homology domain, and 3) Ca(2+) influx-induced translocation of GFP-tagged PKCtheta, as a marker of DAG production and/or nPKC activity. Thus, Ca(2+) influx alone via voltage-dependent Ca(2+) channels can generate DAG, thereby activating cPKC and nPKC, whose activation is structurally independent of Ca(2+).

most extensively studied enzymes in eukaryotic cells. We now know that PKC plays a pivotal role in a myriad of cellular functions. Ten isoforms of PKC have been identified so far and have been classified into three categories based on structural differences in the regulatory domain: conventional PKC (cPKC; PKC␣, PKC␤I, PKC␤II, and PKC␥), novel PKC (nPKC; PKC␦, PKC⑀, PKC, and PKC), and atypical PKC (PKC and PKC) (2,3). The C1 and C2 regions in the regulatory domain are responsible for diacylglycerol (DAG) and Ca 2ϩ binding, respectively. DAG comes mainly from plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis. This is caused by phospholipase C (PLC) activation, following agonist binding to a G protein-coupled receptor. In excitable cells, cytosolic Ca 2ϩ signals are generated either by Ca 2ϩ influx through voltagedependent Ca 2ϩ channels (VDCCs) and/or by Ca 2ϩ release from the endoplasmic reticulum through inositol 1,4,5trisphosphate (IP 3 ) receptors upon binding of IP 3 , the other product of PIP 2 hydrolysis (4,5). DAG and Ca 2ϩ activate the first family of cPKCs that have both the C1 and C2 regions. The second family of the novel PKCs is also activated by DAG, but in a Ca 2ϩ -independent manner because of the absence of the functional C2 region. The third family of the atypical PKCs can be activated by phosphoinositide-dependent kinase 1 in a Ca 2ϩindependent manner (6).
We focused this study on PKC␣ and PKC as representatives of cPKC and nPKC, respectively, to probe further into the mechanisms underlying the Ca 2ϩ signaling-induced activation of cPKC and nPKC. To this end, we employed INS-1 cells, an insulin-secreting cell line established from a rat insulinoma (7), as a model system in which VDCCs are the main pathways for the generation of Ca 2ϩ signals. Key observations from other laboratories have shown that PKC␣ is activated within the physiological Ca 2ϩ concentration range in the presence of both DAG and phosphatidylserine (8) and that depolarizing K ϩ concentrations evoke an increase in the IP 3 concentration in rat pancreatic islets, suggesting PLC-mediated production of DAG (9). Two important questions arise: 1) Can depolarizationevoked Ca 2ϩ influx through the opening of VDCCs activate cPKC? and 2) Can Ca 2ϩ influx also activate nPKC, if the Ca 2ϩ * This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and grants from Takeda Science Foundation, the Yamanouchi Foundation for Research on Metabolic Disorders, the Naito Foundation, the Nissan Science Foundation, and the Suzuken Memorial Foundation. This work was also supported by a Program Grant from the Medical Research Council (United Kingdom). 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. § To whom correspondence should be addressed. Tel.: 81-53-435-2249; Fax: 81-53-435-7020; E-mail: hmogami@hama-med.ac.jp. ‡ ‡ MRC Research Professor. 1 The abbreviations used are: PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; DAG, diacylglycerol; PIP 2 , phophatidylinositol 4,5-bisphosphate; PLC, phospholipase C; VDCC, voltage-de-pendent Ca 2ϩ channel; IP 3 , inositol 1,4,5-trisphosphate; GFP, green fluorescent protein; MARCKS, myristoylated alanine-rich C kinase substrate; -GFP, GFP-tagged; -DsRed, DsRed-tagged; PHD, pleckstrin homology domain; TPA, 12-O-tetradecanoylphorbol-13-acetate; DiC 8 , 1,2dioctyl-sn-glycerol; TRITC, tetramethylrhodamine isothiocyanate; TIRFM, total internal reflection fluorescence microscopy; TEA, tetraetheylammonium; Ach, acetylcholine; ER, endoplasmic reticulum.
influx results in production of DAG? Furthermore, in INS-1 cells, as in normal insulin-secreting cells, glucose-induced oscillations in membrane potential elicit repetitive openings of VDCCs (10). This is responsible for oscillations in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ), which causes pulsatile insulin secretion (11). Ca 2ϩ oscillations play an essential role in exocytotic secretion in neuroendocrine cells (12). It is therefore important to know how depolarization-evoked Ca 2ϩ oscillations are decoded into long term physiological modifications, such as insulin secretion, through PKC activation.
Recent advances in the use of green fluorescent protein (GFP) has allowed us to investigate PKC activity in intact living cells by monitoring translocation of GFP-tagged PKC (13,14). Inactive PKCs are located in the cytosol. Upon activation, following PIP 2 hydrolysis, they translocate from the cytosol to other cellular locations, such as the plasma membrane. By simultaneously measuring the cytosolic Ca 2ϩ concentration and the translocation of GFP-PKC␥ in astrocytes, it has been shown that there is a marked temporal correlation between glutamate-elicited Ca 2ϩ spikes and PKC translocation (15). A current model for activation of cPKC (14) proposes that: 1) the [Ca 2ϩ ] i elevation recruits cPKC to the plasma membrane via the C2 region, 2) the site on the enzyme where the pseudosubstrate inhibitory region in the regulatory domain is occupied at rest is exposed and becomes available for substrate binding, and (3) full activation of the enzyme takes place when DAG tightly tethers the enzyme to the plasma membrane via the C1 region. The sequence of these events suggests that translocation and activation of cPKC may not always correspond. This raises a final question: How do we know when the pseudosubstrate inhibitory region is removed (i.e. when exactly does activation of cPKC take place)?
To address these questions, we monitored translocation of PKC␣-GFP, as markers for cPKC, in response to depolarization-evoked Ca 2ϩ influx through VDCCs in INS-1 cells. The Ca 2ϩ influx resulted in translocation of PKC␣-GFP to the plasma membrane. We also assessed the phosphorylation state of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) (16) as another marker of PKC activity, by monitoring translocation of GFP-tagged MARCKS (MARCKS-GFP) with DsRed-tagged PKC␣ (PKC␣-DsRed). When phosphorylated by PKC, MARCKS translocates from the plasma membrane to the cytosol (17). Translocation of MARCKS-GFP to the cytosol took place as soon as PKC␣-DsRed translocated to the plasma membrane upon stimulation of Ca 2ϩ influx. These results indicate that the Ca 2ϩ influx can generate DAG, because cPKC is activated by Ca 2ϩ and DAG. We showed this in three different ways by demonstrating: 1) Ca 2ϩ influx-induced translocation of GFP-tagged C1 domain of PKC␥, 2) Ca 2ϩ influx-induced translocation of GFP tagged pleckstrin homology domain (GFP-PHD), and 3) Ca 2ϩ influx-induced translocation of PKC-GFP as a marker of DAG production. The depolarization-evoked increase in DAG concentration was estimated from in situ calibration to be 1.90 Ϯ 0.02 M. We have demonstrated for the first time that depolarizationevoked Ca 2ϩ influx can generate DAG, thereby activating cPKC and nPKC. We also observed that MARCKS remained phosphorylated through PKC activation as long as the depolarization-evoked Ca 2ϩ oscillations continued. Our results show that short-lived Ca 2ϩ signals can be transduced via PKC activation into long term phosphorylated MARCKS.

Plasmid Construction
PKC␣-pEGFP, PKC-pEGFP, pEGFP-N2, and pDsRed1-N1 were obtained from Clontech Lab, Inc. (Palo Alto, CA). To attain brighter fluorescence of MARCKS-GFP, the GFP of MARCKS-GFP (17) was replaced with pEGFP-N2. pEGFP of PKC␣-pEGFP was replaced with pDsRed1-N1. A GFP-tagged C1 region of PKC␥ (C1 2 -GFP) was produced from a DNA clone of CKR␥1, which was subcloned into an expression plasmid for mammalian cells, pTB701 (18). A cDNA fragment of PKC␥ for C1 region with an EcoRI site in the 5Ј terminus and a BglII in the 3Ј terminus was produced by PCR using pTB701 as a template. The sense and antisense primers used were 5Ј-TTGAAT-TCGCCATGGTGAAGAGCCACAAGTTCACC-3Ј and 5Ј-TTAGATCT-GTCCACGCCGCAAAGGGAGGG-3Ј, respectively. A PCR product for C1 region of PKC␥ was subcloned into the EcoRI site and the BglII site in GFP containing pTB701 (18). The PCR product was verified by sequencing. A GFP-tagged pleckstrin homology domain of PLC␦1 (GFP-PHD) was donated by Dr. Hirose (Tokyo University, Tokyo, Japan) (19).

Cell Culture and Transfection
INS-1 cells (7), insulin-producing cells, were a gift from Dr. Sekine (Tokyo University).The cells were grown in 100-mm culture dishes at 37°C and 5% CO 2 in a humidified atmosphere. The culture medium was RPMI 1640 with 10 mM glucose supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and 50 M mercaptoethanol. For fluorescence imaging, the cells were cultivated on a coverslip at 50% confluency 2 days before transfection. A plasmid of the GFP-or DsRedtagged proteins was transfected into the cells by lipofection using TransIT TM -LT1 (Mirus, Madison, WI). The experiments were performed 2 days after transient transfection.

Imaging Experiments
Epifluorescence Microscopy-The fluorescence images were captured using a Olympus inverted microscope (60ϫ, water immersion objective, and 60ϫ) equipped with a cooled (Ϫ50°C) coupled charge device digital camera (ORCA-II and ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan) and recorded and analyzed on a Aquacosmos imaging station (Hamamatsu Photonics). The excitation light source was 150 W xenon lamp with a Polychrome I monochromator (T.I.L.L. Photonics GmbH., Planegg, Germany). GFP fluorescence was excited at 488 nm for high time resolution of GFP-tagged PKCs digital imaging. We measured the fluorescence intensity of the GFP (DsRed)-tagged proteins (PKCs, MARCKS, PHD, and C1 domain) in the cytosol of the cell, excluding the nucleus, and/or at the plasma membrane, as a marker of translocation. These values (F) were normalized to each initial value (F 0 ), so that the relative fluorescence change was referred to as the "ratio F/F 0 ." For simultaneous measurements of the relative change in fluorescence intensity of the GFP-tagged PKCs in the cytosol and [Ca 2ϩ ] i , GFP fluorescence was excited at 488 nm, whereas Fura2 was excited at wavelengths alternating between 340 and 380 nm. We put a short pass filter of 330 -495 nm to reduce background fluorescence in the light pass between a dichroic mirror of 505 nm and an emission filter of 535/45 nm band pass. The cells transiently expressing GFP-tagged PKCs were loaded with 2 M Fura2/AM in the standard extracellular solution for 30 min at room temperature. The cells were washed twice and used within 2 h. The Fura2 ratio was calibrated using exposure to 10 M ionomycin and 10 mM Ca 2ϩ or 10 mM EGTA in the Fura2-loaded cells without transfection of the GFP-tagged PKCs. A dissociation constant of 150 nM for Ca 2ϩ and Fura2 at room temperature was used. For simultaneous measurement of relative fluorescence change in intensity of MARCKS-GFP and PKC␣-DsRed using a dual band for fluorescein isothiocyanate and TRITC, GFP fluorescence was excited at 488 nm, whereas PKC␣-DsRed fluorescence was excited at 558 nm. To reduce cross-talk between them, an emission filter wheel was used, and alternate emission filters of 535/45-nm band pass and 605/50-nm long pass were synchronously set with excitation filters for GFP and DsRed.
TIRFM (Evanescent Wave)-To obtain high signal-to-noise ratio over the conventional epifluorescence microscopy (see supplemental figure), we installed a TIRFM unit (Olympus) into the same imaging system mentioned above. The incidental light was introduced from the objective lens for TIRFM (Olympus NA ϭ 1.45, 60ϫ). GFP and Fura Red were excited by a 488-nm laser, and each emitted light was collected through 535/45 and 645/75 nm, respectively. For simultaneous measurement of relative fluorescence change in intensity of C1 2 -GFP and [Ca 2ϩ ] i in the Fura Red-loaded cells, we used a W-view Optics (Hamamatsu Photonics), a branching optics that splits the incident light into using a Dichroic mirror of 550 nm, so that two separate images of the GFP and Fura Red fluorescence can be produced.

Membrane Depolarization Induces Transient Translocation of PKC␣ Following [Ca 2ϩ ] i Elevation-
We first examined the distribution of PKC␣-GFP with the help of high time resolution digital imaging. Fig. 1A shows the rapid and reversible translocation of PKC␣-GFP in response to a depolarizing K ϩ concentration (40 mM), which evoked Ca 2ϩ influx through opening of VDCCs. The relative changes in the fluorescence intensities of PKC␣-GFP in the cytosol and at the plasma membrane are plotted in Fig. 1B, as a function of time. PKC␣-GFP translocated from the cytosol to the plasma membrane, and this can be seen by the reciprocal changes in the two parameters ( Fig. 1B) (n ϭ 6). Thus, either parameter can be used as a marker of PKC␣-GFP plasma membrane translocation. We chose to employ the relative fluorescence change in the cytosol as a marker of translocation. Next, we simultaneously measured [Ca 2ϩ ] i and PKC␣ translocation in Fura2-loaded and PKC␣-GFP-expressing INS-1 cells. As seen in Fig. 1C (20). The peak [Ca 2ϩ ] i was no more than 400 nM. When the same cells were depolarized by the K ϩ channel blocker tetraetheylammonium (TEA) (20 mM), the Ca 2ϩ oscillations became more pronounced (21), and the peak [Ca 2ϩ ] i was in the range of 600 -800 nM ( Fig. 2A) (n ϭ 10). To avoid cross-talk between Ca 2ϩ and GFP signals, exactly the same protocol as in Fig. 2A was applied to PKC␣-GFP-expressing cells without Fura2 loading. No translocation of PKC␣-GFP took place in the standard extracellular solution, whereas oscillatory translocations of PKC␣-GFP started immediately after introduction of the TEA-containing solution (Fig. 2B) (n ϭ 5), suggesting a threshold value of [Ca 2ϩ ] i of more than 400 nM for PKC␣ translocation. The temporal profile of the TEAevoked PKC␣-GFP translocations (Fig. 2, C and D) (n ϭ 5) was similar to that observed in response to the membrane depolarization evoked by a high K ϩ concentration (Fig. 1B).
Depolarization-evoked Translocation of PKC␣ Depends on Ca 2ϩ Influx but Not on Ca 2ϩ Mobilization- Fig. 3A shows that upon removal of external Ca 2ϩ , the TEA-induced Ca 2ϩ oscillations and the PKC␣-GFP translocations were abolished (n ϭ 5), indicating that both are totally dependent on Ca 2ϩ influx. We then tested whether PKC␣ can be fully activated in the presence of DAG at physiological Ca 2ϩ concentrations. Using PKC␣-GFP translocation to the plasma membrane as a marker of activation, short exposure to a combination of TEA and the diacylglycerol analogue DiC 8 (100 M) induced sustained PKC␣ activation, despite the fact that [Ca 2ϩ ] i quickly returned to the steady resting level upon removal of the stimulation (Fig. 3B)  (n ϭ 8). This suggests that even a single TEA-evoked Ca 2ϩ spike (within the physiological Ca 2ϩ concentration range) can fully activate PKC␣ in the presence of a sufficient amount of DAG (8).
To compare the effects of Ca 2ϩ influx and IP 3 -mediated Ca 2ϩ mobilization on PKC␣-GFP translocation, we tested, in the same cells, the actions of TEA and acetylcholine (ACh) (using a supramaximal concentration (100 M), which might produce sufficient DAG to activate PKC␣ (see Figs. 7B and 9A)). As shown in Fig. 4A, the TEA-evoked translocation of PKC␣-GFP was much more substantial than that induced by ACh, although the bulk [Ca 2ϩ ] i elevations produced by the two agents were nearly equal (n ϭ 11). For more accurate evaluation of the cytosolic Ca 2ϩ elevation caused specifically by IP 3 -induced store release, we removed the Ca 2ϩ influx component due to capacitative Ca 2ϩ entry through store-operated Ca 2ϩ channels in the plasma membrane (22,23). This was simply done by removing external Ca 2ϩ during stimulation with ACh (100 M) (Fig. 4B). The result was similar (n ϭ 5) to that shown in Fig.  4A, suggesting that there was little effect of store-operated Ca 2ϩ influx on PKC␣ translocation. Reversing the sequence of events (applying TEA first and then subsequently ACh) gave similar results to those shown in Fig. 4 (data not shown).
PKC␣, Activated by Depolarization-evoked Ca 2ϩ Influx, Can Phosphorylate Its Substrate, MARCKS-One line of evidence has shown that [Ca 2ϩ ] i elevations drive translocation of cPKC to the plasma membrane (13,14). However, we do not know whether cPKC can be activated by depolarization-evoked Ca 2ϩ influx alone. To answer this question, we employed a GFPtagged MARCKS as another marker of PKC activity (17), which is a putative and direct substrate for PKC (16), as well as PKC␣-DsRed. We co-transfected both of them into INS-1 cells. When activated PKC phosphorylates the plasma membraneanchored MARCKS, then phosphorylated MARCKS translocates from the plasma membrane to the cytosol. Thus, simultaneous monitoring of PKC␣-DsRed and MARCKS-GFP allows us to test whether depolarization-evoked Ca 2ϩ influx can activate cPKC. Fig. 5 (A and B) shows translocations of MARCKS-GFP and PKC␣-DsRed induced by TEA, indicating that depolarization-evoked Ca 2ϩ influx can activate PKC␣. Phosphorylated MARCKS only slowly and gradually returned to the plasma membrane (ϳ2.5 min) in contrast to the rapid temporal profile of PKC␣ translocation (ϳ30 s) (Fig. 5B) (n ϭ 6). Ca 2ϩ oscillation-driven translocations of PKC␣ kept MARCKS phosphorylated in the cytosol until termination of the repetitive PKC␣ translocations (Fig. 5C) (n ϭ 8).
Depolarization-evoked Ca 2ϩ Influx Induces Translocation of PKC, Despite the Absence of the Functional C2 Domain for Ca 2ϩ Binding-As seen in Fig. 5 (A and B), we now know that depolarization-evoked Ca 2ϩ influx can activate PKC␣. This observation prompted us to explore whether depolarizationevoked Ca 2ϩ influx can generate DAG, because Ca 2ϩ and DAG are required for activation of cPKC (2). It has been shown that K ϩ -induced membrane depolarization increases IP 3 production in insulin-secreting rat pancreatic islets (9). Taken together, these observations indicate that there should be production of DAG in response to depolarization-evoked Ca 2ϩ influx. To test this hypothesis, we employed PKC-GFP as a marker of nPKC activity as well as DAG production because nPKC is activated by DAG alone in a Ca 2ϩ -independent manner (2). TPA caused a rapid and sustained translocation of PKC-GFP in the complete absence of Ca 2ϩ influx (Fig. 6, A and B) (n ϭ 6). The  simultaneous measurements of PKC-GFP translocation and cytosolic Ca 2ϩ concentration, shown in Fig. 6C, confirm that extracellular Ca 2ϩ is not required for PKC-GFP translocation (n ϭ 4). We then applied a depolarizing K ϩ concentration to PKC-GFP-expressing cells. Fig. 7A clearly shows that this stimulus induced a gradual translocation of PKC-GFP to the plasma membrane, which was reversible upon removal of the high K ϩ solution (n ϭ 12). The amplitude of the translocation induced by K ϩ -induced depolarization was comparable with that elicited by ACh (Fig. 7B). It should be noted that ACh still continued to induce translocation of PKC-GFP after [Ca 2ϩ ] i had returned to the resting level, verifying that 100 M ACh generated enough DAG to sustain the translocation until termination of the stimulus (n ϭ 8) (Figs. 7B and 9B). The amplitude of the TEA-evoked translocation was also comparable with that induced by ACh, and the translocation was not synchronous with the TEA-evoked Ca 2ϩ spikes (Fig. 7C) (n ϭ 8).
Depolarization-evoked Ca 2ϩ Influx Translocates GFPtagged Pleckstrin Homology Domain of PLC␦ 1 (GFP-PHD) and GFP-tagged C1 Domain of PKC␥ (C1 2 -GFP)-To fully corroborate the above evidence that the Ca 2ϩ influx gener-ates DAG and activates PKC, we performed further experiments using GFP-PHD and C1 2 -GFP. First, GFP-PHD allows us to visualize IP 3 production by translocating from the plasma membrane to the cytosol because of the 20-fold higher affinity for IP 3 than for PIP 2 (19), such that we can assess indirectly the simultaneous production of DAG upon PIP 2 hydrolysis by a Ca 2ϩ -dependent PLC activation. As shown in Fig. 8A, depolarization-evoked Ca 2ϩ influx resulted in the relatively transient translocation of PHD-GFP, whereas the translocation was sustained during ACh stimulation (n ϭ 11), indicating DAG production. Second, to directly monitor the plasma membrane DAG levels, we also employed translocation of the C1 2 -GFP as a DAG sensor (14,15) using TIRFM. This has a ϳ10-fold higher signal-to-noise ratio (see "Experimental Procedures") than conventional epifluorescence microscopy in terms of the fluorescent protein translocation near the plasma membrane (for example PKC-GFPs) (15). We loaded the C1 2 -GFP-expressing cells with Fura Red to define the relationship between the [Ca 2ϩ ] i change and DAG production in response to the Ca 2ϩ influx. Depolarization-evoked Ca 2ϩ influx clearly translocated C1 2 -GFP to the membrane (Fig. 9A)  ther Ca 2ϩ -or IP 3 -induced Ca 2ϩ release to the C1 2 translocation (data not shown). As long as both high K ϩ and ACh stimulation continued, C1 2 -GFP remained at the plasma membrane. The translocation induced by Ca 2ϩ influx was inhibited by the PLC inhibitor U73122 (n ϭ 4), suggesting that DAG production is mediated by Ca 2ϩ -dependent PLC activation (Fig. 9D).
In Situ Calibration of Depolarization-evoked Increase in DAG Content-We calibrated the depolarization-evoked increase in DAG concentration in single C1 2 -GFP expressing cells. An extracellular solution containing the DAG analogue DiC 8 was introduced at the end of each experiment, following 40 mM K ϩ stimulation, using TIRFM. Fig. 10 shows a representative experiment where application of three different concentrations of DiC 8 (1, 3 and 10 M) resulted in different quasisteady state levels of C1 2 translocation. Because the concentration of DiC 8 inside the cell, at each level, is thought to equilibrate with that outside the cell, the depolarizationevoked increase in DAG concentration was estimated (from the calibration curves of the three experiments) to be 1.90 Ϯ 0.02 M (mean Ϯ S.D.).

Microdomains of Elevated [Ca 2ϩ ] beneath the Plasma Membrane, but Not Elevation of the Bulk [Ca 2ϩ ] i , Are
Required for cPKC Translocation-We have shown that there is a threshold value of the bulk [Ca 2ϩ ] i at ϳ400 nM for PKC␣ translocation in the insulin-secreting INS-1 cells ( Fig. 2A), which is consistent with data from other laboratories (24). More importantly, we have also demonstrated that Ca 2ϩ influx is a much stronger stimulus for PKC␣ translocation than Ca 2ϩ mobilization from intracellular stores, even when the amplitudes of the induced bulk [Ca 2ϩ ] i elevations are similar (Fig. 4A). This finding suggests that microdomains of elevated [Ca 2ϩ ] beneath the plasma membrane ([Ca 2ϩ ] sub ) generated by Ca 2ϩ influx through VD-CCs may play a pivotal role in cPKC translocation in excitable cells rather than the elevated bulk [Ca 2ϩ ] i , which is also consistent with a recent report (25). In other words, there is a threshold value of [Ca 2ϩ ] sub for translocation of cPKC. In neuroendocrine cells, the estimated value of [Ca 2ϩ ] sub at the mouth of open VDCCs is several micromoles/liter (12,26). This indicates that a local [Ca 2ϩ ] of several micromoles/liter may be required for cPKC translocation. Ca 2ϩ mobilization induced by ACh most likely fails to translocate PKC␣ because the [Ca 2ϩ ] rise at the critical sites is insufficient. This could be due to the distance between the plasma membrane and the IP 3 channels in the ER, combined with the substantial Ca 2ϩ buffering capacity in the cytosol (27). However, it could be argued that Ca 2ϩ mobilization from the ER should result in store-operated Ca 2ϩ entry (22,23) and that ACh stimulation therefore also could be expected to cause local Ca 2ϩ elevation beneath the plasma membrane. Nevertheless, it would appear (Fig. 4B) that the magnitude of Ca 2ϩ influx through store-operated Ca 2ϩ channels is insufficient to generate the threshold level of [Ca 2ϩ ] sub needed. Because the entry sites from store-operated Ca 2ϩ channels would be very close to Ca 2ϩ uptake sites into the ER through the powerful Ca 2ϩ ATPase pumps (23, 28 -31), the net delivery of Ca 2ϩ to the cytosol through store-operated Ca 2ϩ channels may be less than through voltage-gated channels even if both channels have similar ranges of Ca 2ϩ concentrations at the mouths of their pores. They could also possibly be separately located. Our finding that Ca 2ϩ entry through VDCCs is sufficient to cause cPKC and nPKC translocation may be important in relation to the control of glucose-elicited insulin secretion, because it has been shown that the L-type VDCCs and the insulin-containing secretory granules are co-localized (26). Thus, local Ca 2ϩ entry in the secretory domains could induce PKC activation important for stimulation of exocytosis (25).
Ca 2ϩ Influx through VDCCs Is Both Necessary and Sufficient for Activation of cPKC-To fully substantiate that Ca 2ϩ influx through VDCCs is both necessary and sufficient for activation of cPKC, we employed the phosphorylation state of MARCKS as another marker of PKC activity. As seen in Fig. 5B, plasma membrane-anchored MARCKS is turned into phosphorylated MARCKS, thereby moving into the cytosol, as soon as PKC␣ is translocated to the plasma membrane by Ca 2ϩ influx through VDCCs. In INS-1 cells, in which PKC␣ and PKC⑀ are predominantly expressed (32), endogenous cPKC and nPKC may move to the plasma membrane in the same manner as the exogenous examples. Thus, we have provided the first direct evidence showing that single Ca 2ϩ spike-driven translocations of cPKC, whose duration is just 30 s long, enable MARCKS to be phosphorylated. The pseudosubstrate inhibitory region of cPKC has been already removed before the association with MARCKS. The cessation of MARCKS translocation and the return to the prestimulation level is very much slower than that of the cPKC translocation, because of sustained MARCKS phosphorylation. We do not know the exact mechanism, but it might result from the net effect of several factors such as diacylglycerol kinase (17), PKC, or a phosphatase that dephosphorylates MARCKS.
Depolarization-evoked Ca 2ϩ Influx through VDCCs Can Generate DAG and Thereby Activate cPKC and nPKC, Whose Activation Is Structurally Independent of Ca 2ϩ -nPKC, which lacks the functional C2 domain for Ca 2ϩ binding, is activated either by DAG or TPA. This can be seen by the sustained PKC translocation induced by TPA in the absence of external Ca 2ϩ (Fig. 6B). However, depolarization-evoked Ca 2ϩ influx through VDCCs can also induce gradual and continuous nPKC translocation to the plasma membrane during [Ca 2ϩ ] i elevation (Fig. 7A). It is possible that some regions of PKC other than the C1 domain can be associated with the plasma membrane. However, two additional experiments using GFP-PHD and C1 2 -GFP have added further credence to the observations (Fig. 7). First, the Ca 2ϩ influxevoked translocation of GFP-PHD (Fig. 8A) indicates that DAG can be generated upon PIP 2 hydrolysis mediated by a Ca 2ϩ -dependent PLC activation (33), although the amplitude of the PHD translocation may parallel the concentration not of DAG but of IP 3 (19). The translocation of GFP-PHD induced by depolarization was relatively transient, whereas it was sustained during ACh stimulation. This suggests a relatively transient increase in DAG concentration (3). Second, the Ca 2ϩ influx-evoked translocation of C1 2 -GFP (Fig. 9A) as a DAG sensor, directly supports the view that DAG synthesis is induced by depolarization-evoked Ca 2ϩ influx through VD-CCs. The simplest explanation for this surprising observation could be that the Ca 2ϩ influx can initiate DAG generation, by triggering PLC activation, thereby translocating and activating nPKC. The fact that the translocation of the C1 domain did not start until the [Ca 2ϩ ] i had nearly reached its peak (Fig. 9B), taken together with the observation that sustained Ca 2ϩ influx induced by 1 M ionomycin kept C1 2 -GFP at the plasma membrane (data not shown), suggests that there may be a threshold value of [Ca 2ϩ ] sub for DAG synthesis. Conversely, DAG synthesis can be detected by monitoring the C1 domain translocation, if the amplitude of the C1 domain translocation parallels the amount of DAG synthesis. Therefore, we tried to estimate the increase in DAG content with in situ calibration (Fig. 10A), which gave a value of 1.90 Ϯ 0.02 M (mean Ϯ S.D., n ϭ 3). In a report from another laboratory, using a biochemical assay, it was calculated that the amount of accumulated DAG is 13 pmol/10 6 cells at 30 s in plateletderived growth factor-stimulated Balb/c/3T3 cells (34). Given a cell volume of ϳ1 pl, the DAG concentration would be 13 M, which is comparable with our data. As shown in Fig. 5, a DAG concentration of ϳ2 M, induced by depolarizationevoked Ca 2ϩ influx, may be sufficient to ensure that activated PKC can phosphorylate MARCKS. Monitoring of C1 2 -GFP translocation has been the most sensitive way of detecting DAG synthesis beneath the plasma membrane so far (14,15). As shown in Fig. 9A, the C1 domain biphasically translocated to the membrane during high K ϩ stimulation; the first phase was transient, and the second phase was sustained. This indicates continuous production of DAG. DAG synthesis can be mediated by PLC (Figs. 8A and 9D) (33) and/or phospholipase D activated by the [Ca 2ϩ ] i rise and/or PKCs (3,9,35,36). However, neither Ca 2ϩ -nor IP 3 -induced Ca 2ϩ mobilization from the Ca 2ϩ stores is important for DAG synthesis, because of the undiminished magnitude of the depolarization-evoked C1 2 translocation in the thapsigargin-pretreated cells.
Our data also have important implications for cPKC activation. If the amplitude of the nPKC translocation reflects the amount of DAG synthesized, depolarization-evoked Ca 2ϩ influx could translocate as well as activate cPKC by generating [Ca 2ϩ ] sub and DAG. Ca 2ϩ signals per se, such as action potential-induced Ca 2ϩ oscillations, could function as second messengers as well as operate as primary activators of cPKC and nPKC in neuronal, endocrine and muscle cells. In other words, Ca 2ϩ signals and the two PKCs signals may not be segregated in certain conditions. Therefore, the roles of these signals in a myriad of cellular functions may overlap.
Short-lived Ca 2ϩ Signals through PKC Activation Are Transduced into Long-lived Phosphorylated MARCKS; Ca 2ϩ Oscillation-driven Activation of cPKC and nPKC May Modulate Long Term Physiological Phenomena-Our finding that Ca 2ϩ oscillation-evoked activation of both cPKC and nPKC can keep MARCKS phosphorylated (Fig. 5) has important implications for the control of long term physiological phenomena such as insulin secretion (37), long term potentiation (38), the redox state in the mitochondria (39), and the control of gene expression (40,41). We can envisage that, as long as Ca 2ϩ oscillation-driven activation of a first kinase such as PKC continues in a "sinus-like" manner, then the first kinase is maintained in the phosphorylated state, which in turn leads to activation of a second kinase on a time scale of hours or days. In this way, not only Ca 2ϩ oscillations but also Ca 2ϩ oscillation-driven activation of both PKCs may modulate a long term physiological phenomenon. Thus, we should bear in mind that two classes of PKCs can be activated in conditions where Ca 2ϩ oscillations take place.