Dictyostelium protein kinase C-delta-like protein is localized in the cell nucleus

Bid Celf ( 1996) 86, I 03-- I 09 0 Elsevier, Paris

103

Original article

Dictyostelium protein kinase C-delta-like protein is localized in the cell nucleus

Yingcai Wang, Hila Rubin, Shoshana Ravid *

Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel (Received 14 February 1996; accepted 6 May 1996)

Summary - The molecular mechanism whereby protein kinase C (PKC) molecules transduce signals into the cell nucleus is unknown. In this study, we provide evidence that Dictyostelium discoideum contains PKCSlike protein that is localized in the nucleus. The Dic- tyostelium PKCSlike protein has an apparent molecular mass of 76 kDa. This protein is already highly expressed in vegetative Dic- tyostelium cells. The expression level remained constant up to 12 h of development, and sharply decreased after 16 h. The PKCGlike protein is phosphorylated in vivo in response to CAMP and phorbol ester stimulation. Immunofluorescent studies, as well as subcellular fractionation experiments, have indicated that Dictyostelium PKCGlike protein is permanently located in the nucleus. Our results may indicate that PKCGlike protein in Dictyostelium functions as a link between CAMP and the tumor-promoting phorbol esters, and events that take place in the nucleus.

Dktyostefium / protein kinase C-delta / nucleus

Introduction

Protein kinase C (PKC), which is involved in one of the major signal transduction systems, is activated upon exter- nal stimulation of cells by various ligands, including hor- mones, neurotransmitters and growth factors. These exter- nal stimuli increase the level of diacylglycerol (DAG), which functions as a second messenger by binding and acti- vating PKC [3, 21, 221. PKC appears to be the receptor for the tumor-promoting phorbol esters, and shows a broad substrate specificity in vitro [21, 221, suggesting that it has multifunctional catalytic activity. The evidence available to date strongly suggests that some, if not all, of the pleio- tropic actions of tumor promoters are mediated through PKCs [13].

Several isoforms of PKC are found in mammals, showing characteristic patterns of expression in different tissues. Based on their structure, PKCs are classified into three groups. The conventional PKCs, (cPKCs), which include the isoenzymes a, pI, /3lI and x are activated by Ca2+, phospholipids and DAG or phorbol esters. The novel isoforms (nPKCs) which include the isoenzymes 6, E, 77, pL, and 0, do not require Ca2+ for activation. The atypical forms (aPKCs), c, r and & are dependent on phospholipids but not affected by Ca2+, DAG or phorbol esters (for reviews see [7,23]).

PKC shows a broad substrate specificity in vitro [21,22], suggesting that it has multifunctional catalytic activity. PKC is involved in a variety of functions, such as cell growth and gene expression. The protein substrates of PKC that may be responsible for these diverse effects are located throughout the cell: on the plasma membrane, in the cyto-

*Correspondence and reprints

sol, on the cytoskeleton, and in the nucleus (for a review, see [22]). Several nuclear PKC substrates have been identi- fied (for a review, see [5]), the phosphorylation of which changes their DNA-binding or regulatory properties, serv- ing as a link between activation of PKC and regulation of transcription. At present, only few substrates of nuclear PKC are known and we are just beginning to understand the functional consequences of phosphorylation. Nevertheless, it is becoming clearer that nuclear PKC has important functions in the regulation of DNA metabolism and the modula- tion of the properties of DNA regulatory proteins (for a review, see [5]).

The direct activation of PKC by phorbol esters suggests that it is critically involved in growth control. It is widely accepted that PKC plays a pivotal role in the regulation of proliferation and differentiation. As these processes are dependent on the control of nuclear events, it is obvious that PKC growth-regulatory actions must occur either via signals that reach the nucleus after PKC activation, or that PKC must reside in the nucleus. The isoenzyme PKCG has been localized to the cell nucleus (for a review, see [5]). Overexpression of PKCs in CHO cells resulted in cell division arrest [32] and more recently it was found that stimulation of the platelet-derived-growth factor /? receptor activates PKCS [ 171. These findings make PKCG an interesting nuclear signaling protein, for our research.

Although significant progress has been made toward understanding PKC activation and its in vivo substrates, the cellular events following its activation are still not understood. The major reason for this is the difficulty in combin- ing molecular genetics, biochemistry and cell biology in order to successfully address questions concerning signal transmission. Dictyostelium discoideum is an excellent choice for the study of signal transduction mechanisms: it has all the capabilities mentioned above, and, in addition, Dictyostelium has emerged in recent years as an excellent

104 L’ Wang 621 u/

organism for investigation of basic cellular processes ]9$ 241, due in part to the development of gene targeting technology for the organism and new molecular genetic tools IQ

Extracellular CAMP induces Dictyostelium chemotaxis and cell differentiation by a complex sensory transduction mechanism. Binding of CAMP to the cell surface receptors induces formation of 1,4,5-ttisphosphates (IP,) and DAC by hydrolysis of phospholipids by phospholipase C (PLC), and there is also an increase in Ca*+ influx (for review see [3 11). As mentioned above, in mammalian cells the primary effect of DAG is to activate PKC, and is possible that the same mechanism also operates in Dictyostelium. Various lines of evidence have indicated that Dicbyostelium-like mammalian ceils contains several PKCs. Recently, we have isolated a novel PKC that plays an important role in Dic- tyostelium chemotaxis and development [I, 26, 271. Luderus et al [ 181 repotted a PKC-like activity in Dictyoste- lium. Furthermore, phorbol esters, which directly activate mammalian PKC, have been. shown to induce gene expression in DictyosteEium cells, possibly via PKC activation. [l 11. In this paper we report the identification of PKCSlike protein in Dictyostelium and its localization in the nucleus, which suggests that this PKCMilce protein plays a role in signal transduction to the nucleus.

Materials and methods

Cell culture and development

Growth and development in suspension of Dictyostelium discoid- cum strain A x 2 cells was as de&bed [4]. To develop cells on plate, amoebae were harvested from the HL-5 growth flasks at a density of less than 5 x 106 cells /ml, washed free of HL-5 in MES buffer (20 mM MES (pH 6.8), 0.2 mM CaCI,, 2 mM MgSO,), resuspended in the same buffer at~a density of 2 x I07 cells/ml and pIated on plates containing MES buffer + 2% agar.

Reagents

MES, phenylmethybulfonyl fluoride (PMSF), leupeptin, pepstatin, sodium deoxycholate, HI histone, phosphati&Iserine (PS), DAG, phorbol 1 Zmyristate- 13-a&ate. (PMA), StuphylococCus A, and DAPI were purchased from Sigma, and 3*P from Amers- ham. PKCG antibody, anti-phosphotyrosine (anti-Tyr(P)) and the peptide used to raise the antibody were obtained from Santa Cruz Biotechnofogy, and fluoresceinisothiocyanate (FITC)-conjugated, affinity-purified goat anti-rabbit IgG from Jackson.

Analysis of PKCG-like protein during Dictyostelium cell cycle

A x 2 cells were developed on plates as described above. The cells were collected from the plates and at different time points, washed once in ice-cold phosphate buffer and the peiiet resuspended in 20 mM Tris (pH 7.5). 2 mM EDTA and 2 mM EGTA. The cells were Iysed by sonication using ultrasonic cell disruptor (Microson) model XL with smah-sized tip at 50% output power, and the extract was spun in a microcentrifuge for 10 min at 4OC. The samples were then loaded on 7.5% a sodium dodecyl sul- fate-polyacrylamide gels (SDS-PAGE) [14] and subjected to Western blot analysis using PKCS antibody (Santa Cruz Bio- technology).

Preparation of PKC6 antibody3taphylococcus A cell mixture

Staphylococcus A (50 ~1) which had been washed three times in 1 x lysis buffer (see below) plus 1 mdmi BSA was added to 0.5 pg PKCS antibody or 1 pg anti-Tyr(P) and incubated at 4°C on a rotator for at least 30 min prior to the addition of cell lysate.

lmmunoprecipitation

A sample of 2 x 106 cehs was added to an equal volume of ice cold 2 x immunopr~ipitation buffer (2 x IP) (40 n&I Tris {pH 7.5), 0.2% NP40, 2 mM D’IT, 10 mM EDTA, 20 mM NaHSC$. 50 mM sodium pyrophosphate, 2O@mM NaF, 200 mIvi potassium phosphate (pH 7.5), 10 mg/ml KNase) and protease iahib@rs mix (IO0 pg/ml PMSF, 2 &+tl leupeptins and 2 pg/mI pepstatin)., and the extract was spun in a microcentrifuge for tO min at 4°C. The supernatants were then added to preadsorbed-anti-PKGd antibody- or anti-Tyr(P)-Staphylococcus A cell mixture and incubated for 1 h at 4°C with rotation. The samples were-then centrifuged in a microcentrifuge and the pellet was washed three times in 1 x IP buffer with 1 mg/mI BSA, and once with 1 x IP buffer without BSA. The pell.ets were resuspended in SDS-PAGE-&m ple buffer and boiled for 5 min. The supernatants from a microcentrifuge spin were loaded onto SDS-PAGE gels.

Western immunoblotting

A sample of 2 x 107 cells was washed in IO ml ice-cold PBS (pH 7.4) and pelleted and lysed by the addition of 1 ml @e-cold RIPA buffer (PBS (pH 7.5), cont@ing 1% NP40,0.5% sodium deoxy- &orate, 0.1% SDS), with freshly added protease inhibitor mix. The sampIes were incubated on ice for 30 ruin, land cen&uged for 20 min at 4°C. Protein was determined by the method of Peterson [25], and 25 pg of cell Iysates were electrophoresed on 7.5% SDS-PAGE gels. The~Western blots were probed withanti: PKCS or anti-Tyr(P). ECL was performed using a kit-from Amersham Corp.

For peptide neutralization, 0.1 pg of PKCG antibody was mixed with 1 pg of the peptide, which was used to raise the antibody, incubated for 2 h at room temperature or at 4aC overnight. The mixture was used for a Western blot analysis, as described above.

in vivo phosphorylation

A x 2 cells were developed in MES buffer for 4 h, as previousfy described [4]. 5 mM caffeine was added to the cell suspension 30 min prior to addition of 0.1 mCi/ml 3zP. kiter an~addiitional 1 h the cells were stimulated with 1 @f CAMP or 100 n@I PMA, lysed by the addition: of 200 @ RIPA and incubated on ice for 30 min. The samples were mierocentrifuged for IO t&n at 4°C the supernatants were added to preadsorbed PKCGantibody- Staphylococcus A ceIlmixture, prepared as described-above, and incubated for at Ieast I h at 4°C with rotation. The sampIes were spun in a microcenttifuge and washed three times in IP buffer plus 1 mg/mI BSA and once with IP buffer alo~ne. The pellets were resuspended in SDS sample buffer and boiled for 5 min. The supematants from the microcentrifuge spin were-then road& onto SDS-PAGE gels. To determine the relative amount of PKC&Iike protein immunoprecipitated, we-used densitometric scanning, and the amounts of s2P incorporated into PKC&hke protein were determined using a Phosphorhnager. Relative phosphorylation of PKCGlike protein was determined by dividing the values obtained for s*P incorporation by the values obt#ned for the amount of PKCGlike protein immunoprecipitated.

Immunojluorescence staining

Indirect immunofluorescent staining was performed using the agar-overlay technique [ 101. A rabbit polycloaal antiserum directed against PKCS was applied at~l: 100 dilution, foilowed by the application of FITC-conjugated, affinity-purified goat anti- rabbit IgG at I:25 dilution, which had been preadsorbed on Dic- tyostetium cell extract, as previously described [lo], and DAPI staining [28].

For cAMP or PMA stimulation, Dictyostelium cells were placed on and~allowed to attach to a coverslip for 30-m& and the coverslips were then placed in a Petri dish co&&ring. 10. ml MES buffer and 1 @4 CAMP or 100 nM PMA. After incubaiion at.the indicated time points, the coverslips were washed in PBS itnd subjected to immunofluorescent staining as described [lo].

PKC6 from Dictyostelium

Subcellular fracfionation

A x 2 cells were developed in a shaking flask for 4 h as described [4] and stimulated with either 2 @ CAMP or 100 nM PMA. At the indicated time points, 2 x 107 cells were lysed in 1 ml lysis buffer (50 mM Hepes, (pH 7.5),40 mM MgCJ, 20 mM KCl, 0.15 mM spermidine, 5% sucrose, 14 mM pmercaptoetha- nol, 0.2 mM PMSF, 10% percoll and 1% NP40) and incubated for 10 min on ice. The samples were centrifuged at 3000 g for 5 min to pellet the nuclei, the cytosol and the cell membrane remaining in the supematant. The nuclei pellet was resuspended in 1 ml lysis buffer minus NP40 and the unlysed cells were spun down by centrifugation at 150 g for 5 min. The nuclei were pelleted from the supematant by centrifugation at 3000 g for 5 min, and resuspended in 0.5 ml lysis buffer with out NP40 and Per- ~011. Samples of 40 pg of nuclei and cytosol fractions were subjected to Western blot analysis.

Results

Identification of PKC&like protein in Dictyostelium cells

A polyclonal antiserum raised against PKCS peptide corre- sponding to amino acids 657-673 mapping within the car- boxy terminus of PKCG of rat origin recognized a band with an approximate molecular mass of 76 kDa in homogenates of vegetative (fig 1, lane 1) and 4-h developed (fig 1, lane 2) Dictyostelium cells separated by SDS-PAGE. This immu- nostaining was totally prevented by addition of the syn- thetic PKCG peptide, which was used to raise the antibody (fig 1, lanes 3 and 4). A 76-kDa band was also immunoprecipitated from vegetative (fig 1, lane 5) and 4-h developed (fig 1, lane 6) Dictyostelium cells. These results may indicate that Dictyostelium similar to mammalian cells contains PKCG protein (hereafter termed Dictyostelium PKCGlike protein). The molecular mass of Dictyostelium PKCGlike protein (76 kDa) is consistent with previous reports for PKCGisolated from various mammalian cells [2, 15, 191.

To probe the role of PKCGlike protein, we studied the expression pattern of the protein during Dictyostelium development (fig 2). Cells were developed on plates for 24 h, collected at 4-h intervals and subjected to Western blot analysis, as described in Materials and methods. We found that in vegetative DictyosteZium cells, the protein is already highly expressed at a level that remained constant up to 12 h of development. From 16 to 24 h of development the level of expression of PKCGlike protein was sharply decreased by about IO-fold. These results may indicate that PKCGlike protein plays a role in the growth and development of Dictyostelium cells up to the late aggregation stage.

PKCGlike protein is phosphorylated in vivo in response to CAMP and PMA stimulation

CAMP stimulation of developed Dictyostelium cells results in phosphorylation of many proteins [30]. It has been reported previously that PMA stimulation of mammalian cells resulted in phosphorylation of PKCS [16, 291. We therefore studied the possibility that stimulation of Dictyo- stefium with CAMP or PMA affects the PKC6like protein phosphorylation levels. To study the phosphorylation levels of PKCSlike protein in response to CAMP and PMA stimulation, PKCGlike protein was immunoprecipitated from CAMP- or PMA-stimulated 3*P-labeled Dictyostelium A x 2 cells, as described in Materials and methods. Figure 3A shows an image obtained from the PhosphorImager of immunoprecipitated 3*P-labeled PKCGlike protein after stimulation with CAMP or PMA from a typical experiment.

kDa 250-

989

PKCG 4 64-

36-

30-

Fig 1. Immuno blot analysis of PKCGlike protein expressed in vegetative and 1 developed Dictyostefium cell. Lane 1, vegetative

1234 56

Dictyostelium cells; lane 2, 4-h developed Dictyosrelium cells; lanes 3 and 4, addition of PKCG peptide; lanes 5 and 6, immu- noprecipitation of PKCGlike protein from vegetative and developed Dictyostelium cells respectively. Samples (40 pg total protein) were subjected to 7.5% SDS-PAGE and blotted onto nitrocellulose. Immunoblots were stained with anti-PKCG antibodies.

PKCG-

V 4 8 12 16 20 24

Fig 2. Expression of PKCSlike protein during the Dictyostelium life cycle. A x 2 cells were allowed to differentiate on MES plates and harvested as described in Materials and methods. At the indicated time tbe cells were lysed as described in Materials and methods and the samples (40 pg of total protein) were subjected to 7.5% SDS-PAGE and blotted onto nitrocellulose. Immunoblots were stained with anti-PKCG antibodies.

106

A

Time

PKCG

+cAMP +PMA

0,s 5’ 1-O’ 30’

I I 1

0 25 50

Time (set)

I I I I I

-10 0 10 20

Time (min)

30

Fig 3. In vivo phosphorylation of PKC&like protein. A developed cell suspension was labeled with 32P (0.1 mCi/tnl) for 30 mitt, titer which 1 @vl CAMP or 100 nM PhiA was applied. Samples were taken at various @me p@nts and immunopreeipi~ for PKC&&e pto- tein .and subjected to SDS-PAGE eIectrophoresis. A. An imageobtained with the Phosphoibnager of PKC&ike proteioimnrunppre&i- tated from cAUP- and PMA-stimulated 32P-labeled cdls..B, C; Reiative phosphotylatian of PKC6like protein versus time &ftixcAlliIp and PMA stimulation. Relative phosphorylation wiis quantified by densitometty and PhosphorZmaging, as described in Materials a& methods. Values recorded in B and C represerit the means of at least five assays with a range of up to 15%.

Figure 3B, C show the quantitation of these phosphorylation reactions. In response to CAMP stimulation, the PKC!& like protein is transiently phosphorylated, with a maximum phosphorylation peak at about 30 s (fig 3B). The pattern of CAMP-dependent PKCGlike protein phosphoiylation is similar to the phosphorylation that we previously reported for another Dictyostelium PKC [33]. PMA stimulation of Dictyostelium ceils also resulted in PKCG-like protein phosphorylation (fig 3C). In this case the effect of PMA on PKC&-like protein phosphorylation took place much later than that of CAMP. As seen in figure 3C, the maximum PKCS1ik.e protein phosphotylation was obtained after 10 min of stimulation with PMA. Like CAMP--stimulated PKCGlike protein phosphorylation, the PMA-stimulated PKCGlike protein phosphorylation was transient (fig 3C). These results may indicate that the PKC&ike protein is regulated by CAMP and PMA.

We then exammed the possibility that PKCGlike protein was phosphorylated on tyrosine residues as was reported for mammalian PKCG [16]. As shown in figure 4, when lysates from A x2 cells were immunoprecipitated with PKCS antibody and subsequently immunoblotted with anticTyr(P) antibody, a 76-kDa protein was readily detected (fig- 4A). The 76-kDa,proteinwas also observed when lysates of A x 2 Cells were immunaprecipitated with anti-Tyi(P) antibodies followed by anti-PKCG immunoblot analysis (fig 4-B): These results indicate that the Dictyostelium PKCGlike protein is phospborylated on tyrosine residue(s).

Dictyostelium PK&-like protein is located predominantly in the nucleus

Localization of PKC&-like protein in the nucleus of L& tyostelium cells was detected- both by immunofluoresoenee

PKCG from Dictyostelium 107

kDa A tB

250-

P

. ‘..

50-, . . * .

Fig 4. PKC6like protein is phosphorylated in vivo on tyrosine residues(s). A x 2 cells were developed, lysed and immunoprecipitated with anti-PKCG antibodies and immunnoblotted with anti-Tyr(P) (lane A), or immunoprecipitated with anti-Tyr(P) and immunoblotted with anti-PKCG antibodies (lane B).

staining and by subcellular fractionation experiments (figs 5, 6). Immunofluorescence staining of PKC&like protein revealed that it is localized in the cell nucleus of vegetative as well as 4-h developed cells (fig 5). It is not distributed evenly in the nucleus, but appeared to be in small particles (2 to 6), which may indicate that it is localized in the nucleolus (fig 5A, C). To confirm the nuclear localization of PKCGlike protein, Dictyostelium cells were stained with DAPI (fig 5B, D). Stimulation with CAMP (for 1 to 2 min) or PMA (for 10 to 60 min) did not affect the localization of PKCGlike protein (data not shown). The signals were completely eliminated by competition with the peptide against which the antibody was raised (data not shown), which con- firms their specificity.

Subcellular fractionation experiments performed with vegetative (data not shown) as well as developed cells also assessed the expression of PKCGlike protein in the nucleus (fig 6). Purified nuclei and cytosolic fractions, prepared as described in Materials and methods, were analyzed by Western blotting. Expression of the 76-kDa PKCGlike protein was exhibited in the nuclear fractions obtained from vegetative (data not shown) and developed Dictyostelium cells (fig 6). Inclusion of the peptide against which the antibody was raised in this immunoblot analysis completely prevented the detection of PKCG like protein in the nuclear fractions (data not shown). Stimulation of Dictyostelium cells with CAMP or PMA did not affect the localization of PKCG-like protein

(fig 6). These results are consistent with the data obtained from the immunofluorescence studies and further indicate that the Dictyostelium PKCGlike protein is located predominantly in the nucleus.

Discussion

In this study, we report the presence of PKCGlike protein in Dictyostelium cells, which is permanently expressed in the cell nucleus. Localization of PKCSlike protein in the nucleus is shown both by immunofluorescence studies and cell fractionation analysis. These results may have important implications for understanding the role of PKC in transduction of an extracellular signal to the cell nucleus, and the regulation of gene expression.

Dictyosfelium PKCS-like protein is phosphorylated in vivo in response-to CAMP and PMA stimulation. This is the first report showing that stimulation of Dictyostelium cells with PMA results in in vivo phosphorylation of proteins. These results indicate that Dictyostelium, like mammalian cells, responds to phorbol ester stimulation by phosphorylation of proteins. It has been reported that stimulation of various mammalian cells by phorbol esters results in phosphorylation of cellular proteins [9, 12, 201. Among these is PKCS, which has a similar phosphorylation time course [16, 291 to Dicfyosfelium PKCGlike protein. Furthermore, similar to mammalian PKCS [16, 171, the Dictyostelium PKCGlike protein undergoes phosphorylation on tyrosine residue(s). The role of CAMP- and PMA-dependent phosphorylation of PKCGlike protein in Dictyostelium is not understood and is now under investigation. We previously found that stimulation of Dictyoste- lium cells with CAMP results in phosphorylation and activation of another PKC [l, 331. It is possible, therefore, that the CAMP- and PMA-dependent phosphorylation of PKCGlike protein plays a similar role.

Previous reports have indicated the localization of various PKCs to the nucleus or the translocation of PKC to the nucleus in response to phorbol ester stimulation (for review see [5]). In addition, in B lymphocytes, CAMP increased nuclear PKC activity [6]. However, the role of these nuclear PKCs is not understood. Interestingly, PKCG-like protein in Dictyosfelium cells is specifically and permanently present in the nucleus. The staining pattern of small particles in the nucleus may indicate that PKCG-like protein in Dictyosrelium is localized in the nucleolus. PKCS from neuroblastoma x glioma hybrid NG 108-15 was also found to be localized in the nucleoli [2]. Stimulation by CAMP or PMA did not affect the localization properties of Dictyostelium PKC&like protein detected by both immunofluorescence and subcellular fractionation. Nevertheless, PMA affected the phosphorylation levels of PKCGlike protein, suggesting the intriguing possibility that PKC&like protein serves in Dictyostelium as a phorbol ester receptor.

Recently we found that Dictyostelium contains a gene homologue to the mammalian PKCG (Dembinsky and Ravid, unpublished data); this gene is now being character- ized. These findings, along with the findings that the Dic- fyosfelium PKCGlike protein resides permanently in the nucleus, open the way for the determination of the role of this protein in extracellular signal transduction to the nucleus. Dictyostelium provides a unique opportunity to explore this role since it allow us to target genes into its genome which allows the investigation of the in vivo role of PKCG.

f Wang Cl trt

FITC DAR

+cAMP +PMA

Tlh¶E 0” 30” 60” 120” 5’ Jo’

CN CNCNCNCNC

Fig 6. Biochemical localization of PKCGlike protein in Dic- tyostelium cells. A x 2 cells were developed on MES plates and stimulated with either 2 PM CAMP or 100 nM PMA. At the indicated time points, 2 x 107 cells were lysed and fractionated into cytosol and~naclear fractions as described in Materials and methods. Nucleic and cytosol (C) fractions (40 gg) were subjected to Western blot analysis as described in Materials and methods.

?~- Fig 5. PKCGlike protein immunolocaiization in & B@wtetiurn ce& Ceils were allowed to attach [- fb glass eoverslips and were then fized-as B r4bscriW in Mareeriais and methods. Cells were f$&n- s$b&cted to indirect immunofIuo.re.scence “. @aIning with anti-PC6 or DAPI. A-;*. Vegets- ri,‘; &xi Dictyoste3ium cells. C, D, 4-h &v&pad Dii?: ‘= tyihstelium celk.

Acknowledgment This study was supported by grants from the US-Israel Bina- tional Science Foundation and Israel Cancer Association.

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Dictyostelium protein kinase C-delta-like protein is localized in the cell nucleus

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