Rat liver nuclei protein kinase C is the isozyme type II.

Rat liver nuclei protein kinase C is identified as type II isozyme employing monospecific antibodies obtained against each three types of rat brain protein kinase C isozymes. (Yoshida, Y., Huang, F. L., Nakabayashi, H., and Huang, K-P. (1988) J. Biol. Chem. 263, 9868-9873). A major immunoreactive protein band at 80 kDa was revealed by type II isozyme antibodies at each step of purification, nuclear extract included. The nuclear protein kinase C has been purified to apparent homogeneity as revealed by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing a single 80 kDa protein band. It does seem that 66 kDa protein (Masmoudi, A., Labourdette, G., Mersel, M., Huang, F. L., Huang, K.-P., Vincendon, G., and Malviya, A. N. (1989) J. Biol. Chem. 264, 1172-1179) is a major contaminant devoid of any protein kinase activity. The ratio obtained between protein kinase C enzymatic activity over phorbol dibutyrate bound, at various purification steps, indicates that the nuclear enzyme is a phorbol ester receptor. When isolated nuclei were incubated with 12-O-tetradecanoyl phorbol-13-acetate, endogenous protein kinase C activity was elevated about 8-10-fold suggesting the existence of phorbol ester signaling pathway at the level of nucleus. The role of nuclear protein kinase C is delineated in the regulation of inducible gene transcription

Rat liver nuclei protein kinase C is identified as type II isozyme employing monospecific antibodies obtained against each three types of rat brain protein kinase C isozymes.
A major immunoreactive protein band at 80 kDa was revealed by type II isozyme antibodies at each step of purification, nuclear extract included.
The nuclear protein kinase C has been purified to apparent homogeneity as revealed by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing a single 80 kDa protein band. It does seem that 66 kDa protein (Masmoudi, A., Labourdette, G., Mersel, M., Huang, F. L., Huang, K.-P., Vincendon, G., and Malviya, A. N. (1989) J. Biol. Chem. 264, 1172-1179 is a major contaminant devoid of any protein kinase activity. The ratio obtained between protein kinase C enzymatic activity over phorbol dibutyrate bound, at various purification steps, indicates that the nuclear enzyme is a phorbol ester receptor. When isolated nuclei were incubated with 12-O-tetradecanoyl phorbol-13-acetate, endogenous protein kinase C activity was elevated about g-lo-fold suggesting the existence of phorbol ester signaling pathway at the level of nucleus.
The role of nuclear protein kinase C is delineated in the regulation of inducible gene transcription.
One of the major pleiotropic effects of phorbol ester is the activation of protein kinase C' (1). Molecular mechanism of its activation remains far from clear. Recently, considerable interests have been focused on the events activated at the site of nucleus during signal transduction (2). Prominent among these is the elevation of a transcription factor AP-1 upon phorbol ester treatment of cells (3)(4)(5). The exact mechanism of these events is not known at the present moment. However, a positive regulatory role of protein kinase C at the site of  nucleus in cellular transcription (6) seems obvious. It is in this background that we have documented (7) that protein kinase C is located in the rat liver nuclei. The present paper is a logical extension of our recent work (7) and deals with the isozyme nature, phorbol dibutyrate binding, and purification to apparent homogeneity of nuclear protein kinase C. Using monospecific antibodies (8) raised against three types of rat brain cytosolic protein kinase C, it is revealed here that the rat liver nuclei mainly contain protein kinase C isozyme II. The monospecific antibodies obtained against type II isozyme identified 80-kDa immunoreactive protein band at each step of purification. In addition, data presented here indicate that phorbol ester-induced signaling pathway operates at the site of isolated nucleus.  (Fig. 2, lane b). Fractions, shaded (during pH elution), were pooled and served as a source of purified protein kinase C. This fraction revealed a single band at 80 kDa by silver nitrate staining of SDS-polyacrylamide gel electrophoresis (Fig. 2,  After elution with pH the DEAE-cellulose column was further eluted with buffer A supplemented with 120 mM NaCl (Fig. 1). Almost negligible enzymatic activity was seen in these fractions.
The SDS-polyacrylamide gel electrophoresis of the two elutions from the DEAE-cellulose column is shown in Fig. 2. The material eluted with pH and containing protein kinase C activity showed an 80-kDa protein band (Fig. 2, lane a), whereas the 66-kDa protein band (Fig. 2, lane b) was retained on the column during pH elution, which was later eluted with 120 mM NaCl. Table I depicts the protein kinase C enzymatic activity and phorbol dibutyrate binding at each purification step. The enzymatic activity and phorbol dibutyrate binding run in parallel at each step of purification when evaluated either in terms of fold of purification or percentage of recovery. Almost identical ratio was observed, in all the purification steps, between protein kinase C activity over phorbol dibutyrate bound.

Immunoblot
with Three Types of Monospecific Antibodies- Fig. 3 illustrates the immunoblot at each purification step starting from the nuclear extract. The monospecific antibodies against rat brain protein kinase C isozyme type I (Fig. 3A) or type III (Fig. 3C) revealed very faint immunoreactivity in some fractions (lanes a or d). The major immunoreactive 80-kDa protein band was identified by monospecific antibodies against isozyme type II at each purification step (Fig. 3B).
Immunoreactive protein at a lower molecular mass was seen in the enzyme material obtained after phenyl-Superose step (Fig. 3B, lane C). However, this was a nonspecific interaction with immunoglobin unrelated with protein kinase C. Such nonspecific interactions are usually seen (14) and their nature remains enigmatic.
Cofactor Requirements of Nuclear Protein Kinase C-Detailed characteristics of purified (single band at 80 kDa revealed by silver nitrate staining on SDS-polyacrylamide gel electrophoresis) nuclear protein kinase C are listed in Table  II. The enzyme requires calcium plus phosphatidylserine for manifestation of its activity. Relatively higher calcium optimum is sustained in the fully purified preparation. The calcium was replaced by phorbol ester and reasonable kinase C activity was manifested, without any added calcium, in the presence of phosphatidylserine plus TPA or diacylglycerol. However, Ca*+ + TPA (or diacylglycerol) at various calcium concentrations tested did not give any appreciable enzymatic activity. It did appear that there was a slight shift for optimum calcium requirement in the enzyme derived from the final purification step (IInd DEAE) as compared with the penultimate step (Mono Q, Ref. 7). Likewise, appreciable activation of the enzyme by phorbol ester or diacylglycerol over the basal activity (i.e. the activity monitored with Ca*+ + phosphatidylserine) was observed. An activation to such an extent was not seen with contaminating 66-kDa protein (Ref. 7).
Phorbol Ester Induced Signal at the Level of Isolated Nuclei-Isolated rat liver nuclei were incubated with phorbol ester (0.1 or 81 nM) and 6-8-fold rise in the nuclear protein kinase C activity was observed (Table III). Under similar incubations with TPA, Buckley et al. (15) have observed a several hundred-fold rise in protein kinase C activity endogenous to rat liver nuclei. In our hands only &lo-fold activation by TPA could be observed. We have preincubated the isolated rat liver nuclei with 1 mM calcium as carried out by Buckley et al., but even in that condition we have not been able to see more than lo-fold increase in nuclear protein kinase C activity due to phorbol ester-induced signaling.

DISCUSSION
Through the use of a high molarity sucrose (1.3 M) in the homogenization medium and maintaining a higher sucrose molarity (2.2 M) in subsequent suspension and centrifugation procedures, we have isolated nuclei from rat liver devoid of The pooled fraction after II-DEAE chromatography (i.e. eluted by stepwise pH changes) was subjected to these measurements. Protein kinase C activity was assayed as described under "Experimental Procedures." These values are based on four independent determinations for which standard errors were not more than 10%. Standard assay medium contained in a total volume of 100 ~1: 2 mM phenylmethylsulfonyl fluoride, 20 mM Tris-HCl, pH 7.5, 1.2 mM EGTA, 81 nM TPA, or 100 JLM diacylglycerol (DAG), 16.6 pg of phosphatidylserine (PS), 20 rg histone (type 111s SIGMA). 20 UM ATP. Isolated nuclei suspended in a medium containing 0.25 mM sucrose, 25 mM Tris-HCl, pH 7.5, 1 mM MgC& were incubated in 25-ml Erlenmeyer flasks (each set of experiments in quadruplate) with TPA at 37 "C for 3 min. At the termination of incubation each nuclear fraction was centrifuged at 1000 x g for 15 min. The resulting pellet was suspended in a medium containing 2 mM EDTA, 20 mM Tris-HCl, pH 7.5, and 0.5% Triton X-100. It was sonicated six times for 10 s each with a l-min interval in between two sonications and centrifuged at 100,000 x g for 30 min. The supernatant served as the source of protein kinase C. The details of protein kinase C assay are described under "Experimental Procedures," except that the assay medium was supplemented with 100 pM diacylglycerol as recommended (11) due to the presence of more than 0.02% Triton X-100. The protein kinase C activity was found in the nuclei and was partially purified (7).

Additions
One of the major thrusts of this paper has been to get rid of the 66-kDa protein band from the protein kinase C. This has been successfully met with employing a second DEAEcellulose column as a final step. The protein kinase C activity from this column was eluted as a function of pH. The use of classical 120 mM NaCl in Hepes buffer, pH 7.5, did elute the 66-kDa protein band from the DEAE column which was not eluted during pH elution (Fig. 1). Considerable speculation has been attached to the 66 (or 67 kDa) protein fragment usually seen in various kinase C preparations.
According to a section of opinion 66-kDa protein is a proteolytic product of kinase C having lost its calcium-dependent kinase activity. However, it is clearly demonstrated in this paper that 66-kDa protein is a major contaminant, is devoid of kinase activity (calcium dependent or independent), and is not recognized by protein kinase C antibodies.
In fact Parker et al. (16) have documented that the cytosolic protein kinase C has a 67-kDa protein as a major contaminant.
It is indeed surprising that Azhar et al. (17) have attributed protein kinase C activity in the liver to a 64-kDa protein band and have failed to see the 80-kDa protein band in their studies on rat liver enzyme.
A perusal of the purification protocol followed in this paper Furthermore, the ratio between protein kinase C activity over PDBu bound remained identical throughout the purification procedures. This provides strong evidence that the enzyme, under purification, qualified as the receptor of phorbol ester.
Considering cofactor characteristics of the purified nuclear protein kinase C (Table II) one may argue that they are not dissimilar to the partially purified enzyme (7) except that (i) the optimum calcium requirement tends to shift to a lower calcium concentration (2.5 mM instead of 3.5 mM), (ii) the kinase activity elicited (in the absence of added calcium) in the presence of phosphatidylserine + TPA (or diacylglycerol) reached almost identical to the value attained with Ca2+ + phosphatidylserine.
This clearly shows that in the case of nuclear protein kinase C, phorbol ester or diacylglycerol could replace calcium. Thus, the elimination of 66-kDa protein band seems to improve the enzymatic characteristics of the nuclear protein kinase C. Nevertheless, one most important cofactor for nuclear enzyme remains the phosphatidylserine.
In the absence of phosphatidylserine, enzymatic activity was not detected irrespective of the presence of calcium, or phorbol ester, or their combination.
The second major thrust of this paper is that the rat liver nuclear protein kinase C is the type II isozyme (Fig. 3). The isozyme I antibodies or isozyme III antibodies showed minor immunoreactive protein at some of the steps of purification (Fig. 3, A and C). Distinctly enough isozyme II antibodies revealed a major immunoreactive protein band at 80 kDa ( Fig.  3B) at each step of purification starting from nuclear extract up to the final step, i.e. IInd DEAE-cellulose chromatography. It may be recalled that in the rat liver only type II and III protein kinase C isozymes are found. In the cerebellum cells nuclei (18) isozyme type II and type I have been observed, whereas the brain contains all the three types (I, II, and III) of protein kinase C isozymes. These observations do lead to the notion that isozyme II may be responsible for the function of protein kinase C at the site of cell nuclei. It is becoming understandable that each isozyme subserves a different function at various locations within a cell since for the three types the activation kinetics upon stimulation are reported different (19).
Recent studies in rat liver have shown that the protein kinase C endogenous to the nuclei is involved in the signaling pathway initiated by prolactin (20) at the site of the nucleus. Buckley et al. (15) have also shown that there occurs a several hundredfold rise in nuclear protein kinase C activity when isolated rat liver nuclei were incubated with phorbol ester. However, in our hands only 8-lo-fold rise in nuclear protein kinase C activity were seen (Table III) when isolated intact rat liver nuclei were incubated with 0.1 or 81 nM phorbol ester. Irrespective of the discrepancy between our results and that of Buckley et al. (15), it is indeed interesting to be able to initiate a signal by phorbol ester at the site of the nucleus and sustain the nuclear protein kinase C activation. Such an activation provides a tool to address the question of nuclear events associated during signal transduction. The nuclear membrane may not be considered as a wide open frontier passively containing chromatin. It is in fact a hermetic structure with a high selectivity for the molecules passing through it (21). In this context the ATP-stimulated calcium transporting system recently identified in rat liver nuclei is worth citing (22).
The localization in the nucleus of a particular isozyme type of protein kinase C raises questions concerning its role. Based on published observations (5, 23), one obvious role of this enzyme seems to mediate inducible gene expression. The mechanism by which nuclear protein kinase C intervenes the regulation of gene transcription (26) remains to be elucidated.