Expression of cell volume-regulated kinase h-sgk in pancreatic tissue

K. Klingel1, S. Wärntges2, J. Bock2, C. A. Wagner2, M. Sauter1, S. Waldegger2, R. Kandolf1, and F. Lang2

1 Department of Molecular Pathology, Institute of Pathology and 2 Institute of Physiology, University of Tübingen, D-72076, Tübingen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transcript levels of the human serine/threonine kinase h-sgk have been found to be highest in pancreas. In the present study, localization and regulation of h-sgk transcription in pancreatic tissue were elucidated. As was apparent from radioactive in situ hybridization, most pancreatic acinar cells expressed high levels of h-sgk mRNA. h-sgk mRNA-positive cells were also found in ductal epithelia but not in pancreatic islets. In biopsy specimens from patients with pancreatitis, h-sgk mRNA levels were decreased in acinar cells but abundant in numerous mononuclear interstitial cells within areas of pancreatic necrosis and fibrosis. As shown by Northern blotting, h-sgk transcription in DAN-G pancreatic tumor cells is upregulated by osmotic cell shrinkage, serum, phorbol esters (phorbol 12,13-didecanoate), and Ca2+ ionophore A-23187 and decreased by staurosporine and cAMP. In conclusion, h-sgk transcription is regulated not only by cell volume but also by serum, protein kinase C stimulation, cAMP, and increase of intracellular Ca2+ activity. The kinase may participate not only in normal function of exocrine pancreas but also in fibrosing pancreatitis.

serine/threonine kinase; acinar cells; pancreatitis; pancreatic ducts; macrophages


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE IMPORTANT CHALLENGES of cell volume constancy is epithelial transport. Cellular uptake and extrusion of transported ions and organic substances must be coordinated to avoid excessive alterations of cell volume. Similar to other epithelia, pancreatic cells regulate their cell volume by activation of ion transport across the cell membrane (11). The mechanisms linking cell volume to volume regulatory ion transport, however, remain poorly understood. Recently, the putative serine/threonine kinase h-sgk was cloned from Hep G2 cells and shown to be transcriptionally regulated by cell volume (17). The kinase is highly homologous to the serum glucocorticoid-inducible kinase sgk previously cloned from rat mammary tumor cells (20). Expression of h-sgk is markedly increased on hypertonic and isotonic cell shrinkage and is decreased on hypotonic and isotonic cell swelling (17). As shown by Northern blot analysis, the kinase is expressed in all tissues studied, including pancreas, liver, heart, skeletal muscle, placenta, kidney, and brain (17). However, by far the highest transcript levels were observed in the pancreas, pointing to an important role of h-sgk in this tissue (17). Because epithelial transport involves several cell volume regulatory mechanisms (9), the cell volume-sensitive kinase h-sgk, if expressed, could influence the function of the exocrine pancreas. The present study was performed to elucidate the cellular distribution of h-sgk mRNA in normal and inflamed pancreatic tissue. Additional experiments were performed to disclose transcriptional regulation of h-sgk in pancreatic tumor cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In situ hybridization. Tissue specimens of normal pancreas (n = 6) and of pancreas from patients with acute (n = 6) and chronic (n = 6) pancreatitis obtained at routine biopsies were fixed in 4% paraformaldehyde-0.1 M sodium phosphate buffer (pH 7.2) for 4 h and embedded in paraffin. Four-micrometer tissue sections were dewaxed and hybridized basically as described previously (5-7). The hybridization mixture contained either the 35S-labeled RNA antisense or sense control h-sgk probe (500 ng/ml) in 10 mM Tris · HCl, pH 7.4, 50% (vol/vol) deionized formamide, 600 mM NaCl, 1 mM EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.05% bovine serum albumin, 10% dextran sulfate, 10 mM dithiothreitol, 200 µg/ml denatured sonicated salmon sperm DNA, and 100 µg/ml rabbit liver tRNA. Hybridization with RNA probes proceeded at 42°C for 18 h. Slides were then washed as described previously (5, 7) followed by 1 h at 55°C in 2× standard saline citrate. Nonhybridized single-stranded RNA probes were digested by RNase A (20 µg/ml) in 10 mM Tris · HCl, pH 8.0, and 0.5 M NaCl for 30 min at 37°C. Tissue slide preparations were autoradiographed for 3 wk (7) and stained with hematoxylin and eosin.

Transcriptional regulation of h-sgk by cell volume. DAN-G cells (pancreas carcinoma cells, ACC 249) were maintained in RPMI 1640 (GIBCO BRL), 5% CO2, and 10 mM glucose at 37°C, pH 7.4, supplemented with 10% (vol/vol) FCS. Cells were grown to 90% confluence and subsequently homogenized in TRIzol (GIBCO BRL) (~0.4 × 106 cells/sample). Total RNA was isolated as indicated in the protocol provided by the distributor. Northern blots were prepared with 15 or 20 µg of total RNA, each with a separate control that had been electrophoresed through 10 g/l agarose gels in the presence of 2.4 M formaldehyde. Vacuum blotting (Appligene Oncor Trans DNA express vacuum blotter, Appligene, Heidelberg, Germany) was used for transfer of the RNA on positively charged nylon membranes (Boehringer Mannheim, Mannheim, Germany), which were then cross-linked under ultraviolet light (UV Stratalinker 2400, Stratagene, Heidelberg, Germany). Overnight hybridization was performed in DIG-Easy-Hyb (Boehringer Mannheim) at a probe concentration of 25 µg/l at 50°C. The digoxigenin-labeled probe was generated by PCR as described in detail previously (17). For autoradiography, filters were exposed for an average of 5 min to X-ray film (Kodak).

Fluorescence measurements for determination of cell volume. DAN-G cells were grown on glass coverslips as described in Transcriptional regulation of h-sgk by cell volume. Calcein fluorescence intensity was utilized for determination of cell volume changes of single DAN-G cells. To this end, cells were loaded by exposure to 2 µM calcein AM (Molecular Probes) for 15 min and cell volume was measured microfluorometrically by exciting the dye with a 10-µm-diameter spot of light at 497 nm while monitoring the emission at 512 nm. Calcein is insensitive to changes in intracellular pH and Ca2+ (1). Swelling or shrinkage of cells is accompanied by a decrease or increase, respectively, in dye concentration in the cells (14). Therefore, changes in cell volume are expected to be reflected by changes in the fluorescence intensity, i.e., decreased intensity during cell swelling and increased intensity during cell shrinkage. A linear relationship between osmolarity of the perfusion medium and fluorescence intensity has been demonstrated using this technique (1). The relationship is linear because dye concentration is inversely proportional to cell volume and cell volume is inversely proportional to external osmolarity for cells that exhibit osmometric behavior. Fluorescence was measured using a ×100 oil-immersion lens (Zeiss). Fluorescence in the absence of calcein was <1% of the values in the presence of the dye and was not significantly modified by the experimental maneuvers. Measurements were performed at 37°C. The control solution contained (in mM) 114 NaCl, 21 NaHCO3, 5 KCl, 5 glucose, 1.2 CaCl2, 1 Na2HPO4, and 0.8 MgCl2, pH 7.4, equilibrated with 95% O2-5% CO2. To increase osmolarity, 50 mM NaCl or 100 mM raffinose was added to the solution.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Localization of h-sgk mRNA in normal pancreas. To elucidate the mRNA distribution patterns of h-sgk kinase in pancreatic tissue at the cellular level, various tissue specimens of intact pancreas were investigated by in situ hybridization. As demonstrated in Fig. 1A, hybridization of representative pancreatic specimens with the 35S-labeled h-sgk-specific antisense mRNA probe showed a widespread expression pattern of h-sgk kinase mRNA in the exocrine pancreas. Importantly, a considerable variation of levels of transcription was observed in the numerous hybridization-positive acinar cells. In addition to cells of the acini, cells of the pancreatic ductal system were also found to express h-sgk mRNA in differing amounts (Fig. 1B). Moreover, a small number of h-sgk mRNA-positive mononuclear cells were scattered within the connective tissue around the pancreatic ducts (Fig. 1B). No expression of h-sgk mRNA was observed in pancreatic islets. Hybridization with the 35S-labeled sense mRNA for control did not lead to any labeling of pancreatic cells (Fig. 1C).


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Fig. 1.   Visualization of h-sgk mRNA in unaffected pancreatic tissue by radioactive in situ hybridization. High levels of h-sgk mRNA transcripts are detected in the majority of acinar cells (A). In addition to acinar cells, pancreatic ductal cells (arrows) as well as single mononuclear cells situated within the connective tissue around the ducts (arrowheads) are found to express high copy numbers of h-sgk mRNA (B). No autoradiographic signals are observed when pancreatic tissue probes are hybridized with the 35S-labeled RNA sense control h-sgk probe (C).

h-sgk mRNA distribution in pancreatitis. To compare h-sgk transcription patterns of inflamed pancreas to normal tissue, biopsy material was investigated by radioactive in situ hybridization. Figure 2A represents an example of typical hybridization patterns of pancreatic tissue specimens that were obtained from patients with acute pancreatitis. As demonstrated in Fig. 2A, transcription of h-sgk mRNA in acinar cells of affected pancreas is markedly reduced compared with intact tissue. However, h-sgk mRNA was consistently observed in interstitial cells that were interspersed between the acini. In addition, investigation of tissue probes from patients with chronic pancreatitis revealed abundant hybridization-positive mononuclear cells in areas of fibrosis (Fig. 2B).


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Fig. 2.   In situ detection of h-sgk mRNA expression in pancreatic tissue from patients with pancreatitis. Compared with normal exocrine pancreas, acinar cells from affected pancreatic tissues reveal reduced levels of h-sgk mRNA (A). However, numerous hybridization-positive interstitial mononuclear cells are present in injured areas of pancreatic tissue obtained from patients with acute (A, arrowheads) and chronic (B) pancreatitis.

Regulation of transcript levels in DAN-G pancreatic cells. As demonstrated by Northern blot analysis, DAN-G cells transcribe h-sgk depending on ambient osmolarity (Fig. 3). Within 2 h, the transcript levels are enhanced by an increase of extracellular osmolarity caused by addition of either 50 mM NaCl [52 ± 19% (SE), n = 7] or 100 mM raffinose (25 ± 9%, n = 3). Moreover, addition of 10% FCS (59 ± 18%, n = 4), 10 µM Ca2+ ionophore A-23187 (128 ± 61%, n = 4), and 100 nM phorbol 12,13-didecanoate (PDD; 108 ± 26%, n = 9) significantly increased h-sgk transcript levels, whereas 1 µM staurosporine (-63 ± 8%, n = 5) and 1 mM dibutyryl cAMP (-29.2 ± 9%, n = 7) significantly decreased h-sgk transcript levels (Fig. 3). In cells pretreated for 2 h with the protein kinase C inhibitor calphostin C (1 µM), neither 100 nM PDD (17 ± 11%, n = 3) nor 50 mM NaCl (10 ± 4%, n = 3) significantly increased h-sgk transcript levels. The values were significantly different from the respective effects in the absence of calphostin C. 


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Fig. 3.   Regulation of h-sgk transcription in cultured pancreatic cells (DAN-G) as reflected by Northern blot analysis. A: transcript levels in the presence of (from left to right) Ca2+ ionophore A-23187 (10 µM), phorbol ester phorbol 12,13-didecanoate (PDD, 100 nM), staurosporine (1 µM), dibutyryl cAMP (1 mM), NaCl (50 mM), and FCS (10%) compared with control cells. B: transcript levels in untreated cells (control) and cells treated for 2h with PDD (100 nM) or NaCl (50 mM) without or with calphostin C. The transcripts of h-sgk per 20 µg of total RNA are shown. To certify the regular loading of each lane, ethidium bromide-stained 18S RNA is shown. Northern blots are representative for 3-7 similar experiments.

Cell volume measurements. An increase of extracellular NaCl concentration by 50 mM increased calcein fluorescence by 8.6 ± 1.6% (n = 4), reflecting osmotic cell shrinkage (Fig. 4). A similar increase of calcein fluorescence was observed with addition of 100 mM raffinose (8.2 ± 1.6%, n = 6), calcium ionophore 10 µM A-23187 (7.4 ± 1.7%, n = 5), and 1 mM dibutyryl cAMP (5.2 ± 0.6%, n = 6). PDD (100 nM) did not significantly increase calcein fluorescence (0.4 ± 0.7%, n = 5).


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Fig. 4.   Cell volume of DAN-G cells as reflected by calcein fluorescence. Alteration of calcein fluorescence intensity (Delta FI in % of control value) after increased extracellular osmolarity (addition of 50 mM NaCl; A), after application of 1 mM dibutyryl cAMP (B), after increase of intracellular Ca2+ activity by addition of 10 µM Ca2+ ionophore A-23187 (C), and after application of 100 nM PDD (D). An upward deflection (increase of Delta FI) indicates cell shrinkage; a downward deflection reflects cell swelling.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present data demonstrate that h-sgk mRNA is differentially expressed in distinct cell types of the intact pancreas. The highest levels of transcription are observed in acinar cells. Transcripts of h-sgk are also detected in other cell types of the pancreas, such as pancreatic duct cells and mononuclear cells situated around the pancreatic duct. Both acinar cells and pancreatic duct cells are engaged in electrolyte transport, but acinar cells, in addition, secrete large quantities of digestive enzymes (3).

As previously shown for Hep G2 cells and Madin-Darby canine kidney cells (17), h-sgk transcription is highly sensitive to the volume of DAN-G cells. In Hep G2 cells, isotonic cell shrinkage by simultaneous inhibition of Na+/H+ exchange and Na+-K+-2Cl-1 cotransport has been shown to increase the transcription of h-sgk (17). From this observation it was concluded that the transcription of the kinase is sensitive to cell volume rather than osmolarity. Thus a decrease of cell volume under the influence of secretagogues is expected to increase h-sgk transcription. In shark rectal gland, s-sgk transcription has indeed been observed to be enhanced by the secretagogues vasoactive intestinal polypeptide and carbachol (16). In agreement with earlier reports (8, 13), the volume of DAN-G cells is decreased by both cAMP and Ca2+, which are triggered by a variety of hormones. An increase of intracellular Ca2+ in pancreatic cells yielded the expected stimulation of h-sgk transcription, whereas cAMP significantly decreased the mRNA levels. This latter effect cannot be explained by cell swelling, because cAMP is shown to shrink DAN-G cells. Thus the observations point to a cell volume-independent inhibitory influence of protein kinase A on h-sgk transcription. The stimulatory effect of phorbol esters and the inhibitory action of staurosporine and calphostin C suggest protein kinase C-dependent upregulation of h-sgk transcription. A similar stimulating effect of PDD on h-sgk transcription has been observed previously in macrophages (18). The effect of PDD cannot be explained by cell shrinkage, because PDD exerts no significant effect on DAN-G cell volume. In other tissues, stimulation by protein kinase C (4, 19) and serum (9) increase cell volume, although the effect may vary between different tissues and cannot be extrapolated to DAN-G cells. Nevertheless, our observations point to cell volume-independent regulation of h-sgk transcription. Similar to human sgk, the sgk transcription in the rat has been shown to be stimulated by serum [20] and the abbreviation for the kinase (representing serum glucocorticoid-regulated kinase) was actually coined on the basis of its upregulation by serum (20).

The marked expression of h-sgk in pancreatic epithelial cells suggests a functional role of this kinase in the regulation of epithelial transport. Coexpression of h-sgk with the epithelial Na+ channel ENaC leads to a severalfold stimulation of Na+ channel activity (2, 12, 15). As a matter of fact, h-sgk is the most powerful stimulator of ENaC hitherto described. Most likely, the kinase influences further transport systems engaged in cell volume regulation on the one side and in transepithelial transport on the other. The regulation of h-sgk by secretagogues is thus expected to modulate transepithelial transport.

In tissue specimens of patients with pancreatitis, the transcription of h-sgk is decreased in acinar cells. Cell injury, as it occurs in pancreatitis, is frequently paralleled by cell swelling (10), which could then account for the decline of h-sgk transcription. Moreover, the inflammatory process could lead to dedifferentiation of epithelial cells with subsequent loss of cell-specific h-sgk transcription. On the other hand, pancreatitis is characterized by a marked increase of h-sgk transcript levels in interstitial cells within areas of pancreatic damage. It appears that the increased abundance of h-sgk is caused by both an increased number of h-sgk-expressing cells and increased transcript levels in those cells. Regarding the nature of h-sgk mRNA-expressing cells, it is likely that, because of the typical morphology, the majority of these cells represent macrophages. Indeed, h-sgk transcription is high in macrophages and can be further stimulated in those cells by cytokines such as transforming growth factor-beta 1 (18).

In conclusion, the present paper reveals a distinct pancreatic tissue distribution of h-sgk mRNA, with the most prominent transcription in acinar cells. Inflammatory disease of the pancreas leads to a profound redistribution of h-sgk mRNA. Northern blots reveal the expected sensitivity of h-sgk transcription to osmotic cell shrinkage and increased intracellular Ca2+ activity but reveal apparently cell volume-independent transcriptional regulation by cAMP and protein kinase C.


    ACKNOWLEDGEMENTS

The skilled technical support of Petra Barth, Carmen Ruoff and Sandra Bundschuh is gratefully acknowledged. The authors are indebted to Tanja Loch for secretarial help during preparation of the manuscript.


    FOOTNOTES

This study was supported by the Deutsche Forschungsgemeinschaft grant (No. La 315/4-3), the FORTÜNE program of the Medical Faculty of Tübingen (Nr. 302), the Federal Ministry of Education, Science, Research and Technology, and the Interdisciplinary Center for Clinical Research Tübingen (HIZKF 01 KS 9602).

Address for reprint requests and other correspondence: F. Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany (E-mail: florian.lang{at}uni-tuebingen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 February 1999; accepted in final form 23 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvarez-Leefmans, FJ, Altmirano J, and Crowe WE. Use of ion-selective microelectrodes and fluorescent probes to measure cell volume. Methods Neurosci 27: 361-391, 1995.

2.   Chen, SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96: 2514-2519, 1999[Abstract/Free Full Text].

3.   Cook, DI, and Young JA. Function of the exocrine pancreas. Comprehensive Human Physiology 2: 1327-1343, 1996.

4.   Dausch, R, and Spring KR. Regulation of NaCl entry into Necturus gallbladder epithelium by protein kinase C. Am J Physiol Cell Physiol 266: C531-C535, 1994[Abstract/Free Full Text].

5.   Hohenadl, C, Klingel K, Mertsching J, Hofschneider PH, and Kandolf R. Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization. Mol Cell Probes 5: 11-20, 1991[ISI][Medline].

6.   Kandolf, R, Ameis D, Kirschner P, Canu A, and Hofschneider PH. In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease. Proc Natl Acad Sci USA 84: 6272-6276, 1987[Abstract].

7.   Klingel, K, Hohenadl C, Canu A, Albrecht M, Seemann M, Mall G, and Kandolf R. Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation. Proc Natl Acad Sci USA 89: 314-318, 1992[Abstract].

8.   Kopelman, H, Gauthier C, and Bornstein M. Antisense oligodeoxynucleotide to the cystic fibrosis transmembrane conductance regulator inhibits cyclic AMP-activated but not calcium-activated cell volume reduction in a human pancreatic duct cell line. J Clin Invest 91: 1253-1257, 1993[ISI][Medline].

9.   Lang, F, Busch GL, Ritter M, Voelkl H, Waldegger S, Gulbins E, and Haeussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247-306, 1998[Abstract/Free Full Text].

10.   Lang, F, Busch GL, and Gulbins E. Physiology of cell survival and cell death: implications for organ conservation. Nephrol Dial Transplant 10: 1551-1555, 1995[ISI][Medline].

11.   Muallem, S, and Loessberg PA. Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II Regulation of H+ and HCO3- transporters by Ca2+-mobilizing agonists. J Biol Chem 265: 12813-12819, 1990[Abstract/Free Full Text].

12.   Naray-Fejes-Toth, A., Canessa C, Cleaveland ES, Aldrich G, and Fejes-Toth G. Sgk is an aldosterone-induced kinase in the renal collecting duct: effects on epithelial Na+ channels. J Biol Chem 274: 16973-16978, 1999[Abstract/Free Full Text].

13.   Sweezey, NB, Gauthier C, Gagnon S, Ferretti E, and Kopelman H. Progesterone and estradiol inhibit CFTR-mediated ion transport by pancreatic epithelial cells. Am J Physiol Gastrointest Liver Physiol 271: G747-G754, 1996[Abstract/Free Full Text].

14.   Tauc, M, Le Maout S, and Poujeol P. Fluorescent video-microscopy study of regulatory volume decrease in primary culture of rabbit proximal convoluted tubule. Biochim Biophys Acta 1052: 278-284, 1990[ISI][Medline].

15.   Wagner, CA, Ott M, Melzig J, Wild K, Moschen I, Waldegger S, and Lang F. Cell volume regulated and aldosterone induced kinase hSGK activates the epithelial Na+-channel (ENaC) by enhancement of surface expression and modification of pharmacology. Kidney Blood Pres Res. 22: 363-364, 1999.

16.   Waldegger, S, Barth P, Forrest JN, Jr, Greger R, and Lang F. Cloning of sgk serine-threonine protein kinase from shark rectal gland---a gene induced by hypertonicity and secretagogues. Pflügers Arch 436 (4): 575-580, 1998[ISI][Medline].

17.   Waldegger, S, Barth P, Raber G, and Lang F. Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 94: 4440-4445, 1997[Abstract/Free Full Text].

18.   Waldegger, S, Klingel K, Barth P, Sauter M, Lanzendörfer M, Kandolf R, and Lang F. h-sgk serine-threonine protein kinase gene as early transcriptional target of TGF-beta in human intestine. Gastroenterology 116: 1081-1088, 1999[ISI][Medline].

19.   Wang, WJ, Acs P, Goodnight JA, Giese T, Blumberg PM, Mischak H, and Mushinski JF. The catalytic domain of protein kinase Cdelta in reciprocal delta  and epsilon  chimeras mediates phorbol ester induced macrophage differentiation of mouse promyelocytes. J Biol Chem 272: 76-82, 1997[Abstract/Free Full Text].

20.   Webster, MK, Goya L, Ge Y, Maiyar AC, and Firestone GL. Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13: 2031-2040, 1993[Abstract].


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