Leukemia Research Vol. 12, No. 1, pp. 71-80, 1988. 0145-2126/88 $3.00 + 0.00 Printed in Great Britain. Pergamon Press plc

S P E C I F I C P H O S P H O R Y L A T I O N O F 2 2 - k D P R O T E I N S B Y

V A R I O U S I N D U C E R S F O R G R A N U L O I D D I F F E R E N T I A T I O N I N

M Y E L O I D L E U K E M I C C E L L S

MASAHIRO YAMAMOTO, JUNJI NISHIMURA, HIROSHI IDEGUCHI and HIROSHI IBAYASHI

Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan

(Received 3 June 1987. Revision accepted 5 October 1987)

Abstract--We studied the changes of protein phosphorylation in human leukemic cells by granuloid inducers, using two-dimensional electrophoresis. The phosphorylation of 22 kD, pI 6.0 and 5.8 proteins (pp22) in HL-60 cells or myeloid leukemic cells from patients, was enhanced by treatment with granuloid inducers such as retinoic acid, dimethyl sulfoxide or G-CSF, in common with prostaglandin E2 and theophylline, or dibutyryl c-AMP, which increased intracellular c-AMP. In contrast, pp22 phosphorylation was not induced by the monocytes/macrophages inducer in HL-60 cells, or by the granuloid inducers in lymphoid cells. This phosphorylation occurred within 30 rain and continued for more than 48 h. These pp22 proteins were present in the cytosol and phosphorylated on the serine residues. We now present a possibility that granuloid differentiation in myeloid cells is closely linked with these pp22 phosphorylation.

Key words: Protein phosphorylation, human leukemic cells, granuloid differentiation, c-AMP dependent protein kinase.

INTRODUCTION

PROTEIN phosphorylation plays an important role in the transduction of exogenous signals. Growth fac- tors stimulate tyrosine kinases, which phospho- rylate proteins or enzymes, including the receptors themselves and transmit signals [1-3]. Agents such as tumor promoting phorbol ester (12-o-tetradecanoyl phorbol-13-acetate, TPA) modulate cellular metab- olism with activation of calcium and phospholipid dependent protein kinase (C-kinase), and induce dif- ferentiation into monocytes/macrophages in human promyelocytic leukemia cell line, HL-60 [4, 5]. Cyclic

Abbreviations: c-AMP, cyclic adenosine monophosphate; A-kinase, c-AMP dependent protein kinase; C-kinase, calcium-phospholipid dependent protein kinase; PG, prostaglandin; DMSO, dimethyl sulfoxide; RA, retinoic acid; TPA, tumor promoting phorbol ester(12-o-tetradecanoyl phorbol-13-acetate); G-CSF, granulocyte-colony-stimulating factor; dibutyryl c-AMP, Nr-2'-o-dibutyryladenosine 3~:5'-cycli6 monophosphate; pl, isoelectric point; pp22, phosphorylated proteins with a molecular weight of 22,000 daltons and pI 5.8 and 6.0.

Correspondence to: Dr M. Yamamoto, Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.

71

adenosine monophosphate (c-AMP) is a represen- tative second messenger of signals in various cells, and stimulates c-AMP dependent protein kinase (A- kinase). Many enzymes serving as substrates are phosphorylated by A-kinase to be stimulated or inhibited for the control and modulation of cellular metabolic processes. In HL-60 cells, agents elevating intracellular c-AMP levels, induce granuloid dif- ferentiation [6-8]. On the other hand, the compounds such as retinoic acid (RA), dimethyl sulfoxide (DMSO) or granulocyte-colony-stimulating factor (G-CSF) also lead granuloid differentiation in HL- 60 cells [9-13]. However, these inducers, such as RA or DMSO, do not increase intracellular c-AMP [6]. It is possible that the common pathway stimulated both by c-AMP and by DMSO or RA is related to signal transduction of granuloid differentiation.

Therefore, we attempted to examine the manner in which proteins are phosphorylated in switching from proliferation to differentiation in myeloid cells, particularly along the granuloid pathway, using both agents. We analysed phosphoproteins by two dimen- sional gel electrophoresis and found that DMSO and RA as well as the agents which elevate intraceUular c-AMP commonly stimulate the specific phos- phorylation of 22-kD proteins.

72 M. YAMAMOTO et al.

M A T E R I A L S AND M E T H O D S Materials

DMSO and theophylline were purchased from Wako Pure Chemical Industries Co. (Osaka, Japan). RA, TPA and Nt-2'-o-dibutyryladenosine 3':5'-cyclic mono- phosphate (dibutyryl c-AMP) were from Sigma Chemical Co. (St Louis, MO). Phenylmethylsulfonylfluorid (PMSF) was purchased from Boehringer Mannheim Biochem. (West Germany). Prostaglandin E2 was obtained from Ono Pharmaceutical Co. (Osaka, Japan). 1,25-dihydroxy vitamin D3 and human purified G-CSF were from Chugai Pharmaceutical Co. (Tokyo, Japan). (150 U/ml of G-CSF is sufficient for colony formation of human bone marrow ceils.)

Cells HL-60 ceils were maintained in 10% fetal calf serum

(FCS, from Boehringer Mannheim Biochem.) and Iscove's modified Dulbecco's medium (IMDM, from GIBCO Lab- oratories, NY). Fresh leukemic cells were obtained by preparation with lymphocyte separation medium (Litton- Bionetics, Kensington, MD) from peripheral blood of leu- kemic patients, in which more than 90% of the white blood cells were leukemic.

Two-dimensional electrophoresis of phosphorylated proteins

Cells (1 x 107) were washed twice with phosphate-free Hanks' balanced salt solution (phosphate-free HBSS), resuspended in 1 ml of phosphate-free I-IBSS containing 50 IxCi/ml 32po 4 (Japan Atomic Energy Research Insti- tute, Tokyo, Japan) and incubated at 37°C for 3 h. After washing twice with phosphate-free HBSS, the cells were resuspended in 100 p.l of phosphate-free HBSS. After incu- bation at 37°C with inducers for indicated time, the cells were solubilized in 1% Nonidet P-40 (NP-40) with 1 mM PMSF and crude nuclear residues were removed by centrifugation at 6000 x g for 1 min. Phosphorylated pro- teins (100 ~tg) in the supernatant were finally dissolved in isoelectrofocusing lysing solution containing 9M urea, 2% NP-40, 2% ampholine (pH3.5-10, LKB Bromma, Sweden) and subjected to two dimensional NaDodSO4 polyacrylamide gel electrophoresis, as described by O'Farrell [14]. The second dimensional electrophoresis was carried out on 12.5% polyacrylamide gel. The gels were stained with Coomassie blue, destained, dried and exposed to Kodak X Omat S films (Rochester, NY) for autoradiography.

Subfractionation of cells After preincubation with 3 2 p o 4 and treatment with

inducers at 37°C for 30 min, HL-60 cells (2 x 107) were suspended in 0.5 ml of 0.2 M sucrose containing 20 mM Tris (pH7.4), 3mM CaCI2, and 0.2mM PMSF, then immediately sonicated at 4°C for 15 s, and the crude nuclear residues and unbroken cells were removed by centrifugation at 800 x g for 5 rain. The crude membrane fraction was obtained by centrifugation at 70,000 x g for 60 rain. The proteins in the supernatant (cytosol fraction) was precipitated by 10% trichloroacetic acid and subjected to the two dimensional electrophoresis.

Phospho-amino acid analysis Phosphorylated proteins were extracted from gels as

described [1], and then were partially hydrolyzed in 6 M

HCI at 110°C for 2 h. Phospho-amino acids were analysed by the thin-layer electrophoresis at pH 3.5 (acetic acid/ pyridine/H20, 1:10:189) for 40min at 450V on 10 x 10 cm cellulose thin-layer plates (Funakoshi Pharma- ceutical Co., Tokyo, Japan). The phospho-amino acids were identified by comparison with standards by ninhydrin staining, and autoradiography was carried out.

RESULTS

Stimulation of phosphorylation of common proteins, pp22 by oarious inducers in HL-60 cells

Firstly, we compared phosphorylation patterns in HL-60 cells stimulated by three different compounds: (1) 100 nM PGE2 and 0.5 mM theophylline, which elevate intracellular c-AMP levels and induce gran- uloid differentiation in HL-60 cells [6]. (2) 150 U/ml human purified G-CSF, a physiological stimulator for granulopoiesis in vioo, which also induce granuloid differentiation in HL-60 cells [12, 13]. (3) 5 #M RA, a potent inducer for granuloid differentiation. Little is known about the molecular events in this process. These agents induced phosphorylation or dephosphorylation of some individual proteins, but they commonly enhanced the phosphorylation of two proteins, molecular weight 22kD, pI 6.0 and 5.8 (pp22, shown in Fig. 1), as determined by two dimen- sional electrophoresis. Moreover, the addition of 1.5% DMSO, a polar organic compound, or 0.5 mM dibutyryl c-AMP, a cell-permeable c-AMP analogue which produces in a sustained rise in intracellular c-AMP, also induced the phosphorylation of these proteins (Figs 2B, E). However, neither the treat- ment with 50 nM TPA (Fig. 2F) nor with 100 nM 1,25-dihydroxy vitamin D3 (data not shown), which induces HL-60 cells to form monocytes/ macrophages, enhanced the pp22 phosphorylation. The phosphorylation increased within 5 min after the addition of granuloid inducers, reached a peak around 15-30 min and continued for 48 h after induc- tion (Fig. 2C). Among these agents, high con- centration of DMSO (more than 5%) was a most potent stimulator for the pp22 phosphorylation (Fig. 2D). Thus, we used 15% DMSO for the further analysis of these phosphorylated proteins, although it is toxic for the cells.

pp22 phosphorylation in fresh myeloid leukemic cells The pp22 phosphorylation on the two dimensional

gel electrophoresis was analysed using fresh leukemic cells obtained from our patients. Figure 3 shows that pp22 phosphorylation was enhanced by the treatment with DMSO in blasts from a A ML patient (case 2 in Table 1). In the myeloid leukemic cells from another patient, pp22 were also phosphorylated by DMSO (Table 1). However, in the lymphoid leukemic cells

6 -

0 -

2 1 -

i

9 0 K -

4 5 -

3 0 -

2 1 -

A M W -

9 0 K -

p l l I 7 . 0

M W -

9 0 K -

5 -

0 -

1 -

m

9 0 K -

5 -

0 -

1 -

B

iti w

I I I p lJ I I I 6.0 5 .0 7 .0 6 .0 5 .0

FIG. 1. Effects of PGE2 theophylline, RA and G-CSF on protein phosphorylation in HL-60 cells. HL-60 cells were preincubated with 32po 4 and treated with inducers for 30min. Thereafter, the cells were lysed and the phos- phorylated proteins were resolved on two-dimensional electrophoresis. The second dimension was carried out with molecular weight standard (90, 45, 30, 21 kD). (A) control; (B) 100 nM PGE2 and 0.5 mM theophylline; (C) 150 U/ml G-CSF; (D) 5 o.M RA. Arrows indicate pp22.

73

A

B

C

D

E

I=

•

I?,~ ":''.~ -..* i " -

~ . ~ ~,-

FIG. 2. Effects of inducers on pp22 phosphorylation in HL-60 cells. I-IL-60 cells were prelabeled with 32po 4 and treated with inducers for indicated time, and the pp22 phosphorylation was analysed, as described in materials and methods (A, B, D - F ) . (A) control. (B) 1.5% DMSO 30 min. (C) After incubation in IMDN with 1.5% DMSO and 10% FCS for 48 h, cells were washed with phosphate- free HBSS, labeled with 32po 4 for 3 h and not treated with inducers. The pp22 phosphorylation was analysed as previously described. (D) 15% DMSO 30 min. (E) 0.5 mM dibutyryl c-AMP 30 min. (F) 50 nM TPA 30 min. Arrows

indicate pp22.

74

MW i

| 0 K -

4 5 -

3 0 -

21 -

(A)

|OK-

4 5 -

30 -

2 1 -

p l I I i i '~ 7.0 6.0 5.0

FIG. 3. Effects of DMSO on pp22 phosphorylation in fresh myeloid leukemic cells. The cells were obtained from a patient with AML (Case 2 in Table 1). The pp22 phos- phoryFation was analysed with two dimensional elec- trophoresis. (A) control; (B) treated with DMSO for

30 min. Arrows indicate pp22.

(B)

75

M W

45K--

3 0 - -

21- -

45K--

3 0 -

2 1 -

. . • : - ~ .

pi l l I l 7.0 6.0 5.0

FIG. 4. Detection of pp22 with subfractionation in HL-60 cells. After preincubation with 32po 4 and treatment with DMSO for 30 min, the cells were subfractionationed as described, and analysed by two dimensional electro- phoresis. (A) cytosol fraction; (B) membrane fraction.

Arrows indicate pp22.

76

A -.,--- Pi

" - - - S E R

THR

TYR

"-,-~ OR IGIN

FiG. 5. Phosphoamino-acid analysis of pp22. The phos- phorylated pp22 in HL-60 cells were extracted from the two dimensional electrophoresis gel, partially hydrolyzed in 6 M HC1 at 110 ° for 2 h, and analysed by thin-layer electrophoresis at pH 3.5 (acetic acid/pyridine/H20) for 40 min at 450V. (Pi, free 32po4; SER, phosphoserine; THR, phosphothreonine; TYR, phosphotyrosine, (A)

pI 5.8; (B) pI 6.0.)

77

Protein phosphorylation and granuloid differentiation 79

TABLE 1. pp22 PHOSPHORYLATION BY D M S O IN FRESH MYELOID LEUKEMIC CELLS

Cell pp22 phosphorylation by DMSO

HL-60 + MOLT-4 Case 1 (AML, MI*) -+

2 (AML, M2*) + 3 (AML, M2*) + 4 (AML, M3*) --- 5 (AML, M3*) 6 (CMLt) 7 (T-ALL)

* The classification of acute myelogenous leukemia by French-American-British Co-operative group (1978).

t Chronic phase. The phosphorylation of pp22 by DMSO was analysed by

two dimensional electrophoresis.

from a patient or a lymphoid cell line (MOLT-4) and in the cells from a CML patient in chronic phase, the pp22 proteins were not phosphorylated by the treatment with DMSO. These observations sug- gested that the pp22 phosphorylation was a phenom- enon closely related to immature myeloid ceils.

Subcellular localization and phospho-amino acid analysis of pp22

Two dimensional electrophoresis after subfractionation of DMSO-treated HL-60 cells revealed that these phosphorylated proteins were present in the cytosol fraction, but not in the mem- brane fraction (Fig. 4). The serine residues of pp22 were phosphorylated by DMSO, like the other A- kinase substrates, by analysis of the thin-layer electrophoresis after partial hydrolysis in 6 M HCI of pp22 extracts from the two dimensional gel (Fig. 5).

DISCUSSION

Protein phosphorylation plays an important role in the differentiation in myeloid cells and has been analysed by two dimensional electrophoresis, especially in case of differentiation into monocytes/ macrophages [15-17]. TPA induced phosphorylation of proteins with molecular weight 17 and 27 kD [15, 16] and ten various proteins [17]. We demon- strate in the present report specific phosphoryl- ation of pp22 proteins only by the treatment with inducers for granuloid differentiation, but not for monocytes/macrophages " differentiation. With respect to molecular weight and pI, the pp22 in the present report are unlike the phosphorylated proteins heretofore reported [15-17]. The pp22 were phosphorylated not only in HL-60 cells but also in various myeloid leukemic cells in the very early stage

after the addition of granuloid inducers of different kinds, thereby suggesting that the pp22 phos- phorylation is one of the initial events in the granuloid differentiation. However, since the most potent stimulator for the pp22 phosphorylation was high concentration of DMSO which is toxic for the cells, there remains a possibility that the pp22 phos- phorylation is an epiphenomenon unrelated to the differentiation.

We showed that pp22 were localized in the cytosol and phosphorylated in the serine residues. And pp22 were phosphorylated by agents such as PGE2 and theophylline, or dibutyryl c-AMP which increase intracellular c-AMP [6], suggesting that pp22 are substrates for A-kinase. C-AMP plays a regulatory role not only in granuloid differentiation of myeloid leukemic cells [6-8] but also in normal granuloid hemopoiesis [18, 19]. Although the elevation of intra- cellular c-AMP in HL-60 cells is absent in case of the treatment with DMSO or RA [6], these compounds were shown to modulate A-kinase activity [20-22]. However, it was also reported that RA and DMSO activated c-AMP independent protein kinases, including C-kinase [20, 23, 24]. It is unlikely that the pp22 phosphorylation is catalyzed by C-kinase because a sufficient amount of TPA, a potent acti- vator of C-kinase, showed no increase in the phosphorylation.

Although RA is used for therapy in AML patients and induced differentiation in otoo [25], RA or DMSO rarely induces in-oitro differentiation in fresh myeloid leukemic cells [26]. Unlike HL-60 cells, DMSO or RA also did not induce terminal dif- ferentiation in fresh myeloid leukemic cells in this study (data not shown), but the pp22 phosphorylation occurred in some of them by the treatment of DMSO. While the physiological function of the pp22 proteins are unclear, our results suggest that protein kinases are involved in regulation of leukemic differen- tiation. Thus we can anticipate that further studies on the phosphorylation may lead to a better under- standing of the differentiation in normal and leu- kemic cells.

Acknowledgements--This work was supported in part by grants from the Yamanouchi Foundation of Metabolism and Disease and from the Fukuoka Cancer Society.

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