Cellular Signalling Vol. 7, No. 4, pp. 423429 , 1995. Elsevier Science Ltd

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E C T O P R O T E I N K I N A S E A C T I V I T I E S O N N O N - D I F F E R E N T I A T E D A N D

D I F F E R E N T I A T E D U - 9 3 7 C E L L S

TAREKEGN GEBERHIWOT and GORAN SKOGLUND*

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S- 171 77 Stockholm, Sweden

(Received 27 September 1994; and accepted 21 October 1994)

Abstract--Incubation of intact U-937 cells with 1 ~tm [7-32P] ATP resulted in rapid (10 min) incorporation of radioactivity into phosvitin, kemptide and protein kinase C (PKC)-peptide. The amount of incorporation was depen- dent on substrate type and concentration, and on incubation time. Staurosporine, H-7 and Mg>-exclusion abolished phosphorylation of kemptide and PKC-peptide but not phosvitin. Cyclic AMP and phorbol ester enhanced kemptide and PKC-peptide phosphorylation. Protein kinase inhibitor (PKI) inhibits only kemptide phosphorylation. Cell dif- ferentiation enhanced 2-fold the phosphorylation of phosvitin and PKC-peptide without significant effect on kemp- tide phosphorylation. ATP concentrations sufficient to trigger changes in intracellular Ca 2+ were sufficient to sup- port extracellular phosphorylation reactions. The results suggest the presence of at least three ectokinase activities on U-937 cells that may play important roles in regulating membrane associated specific functions of developing and mature monocytes.

Key words: Protein phosphorylation, ectoprotein kinase, U-937 cells, cell differentiation.

INTRODUCTION

Protein phosphorylation catalysed by protein kinase (PK) serves important regulatory functions in all cells and participates in the control of virtu- ally all aspects of biological life [1, 2]. Protein kinases are distributed throughout the cells, both extra- and intracellularly, but most studies to date deal with intracellular kinases, while extracellular kinases are far less characterized. Recent studies indicate that the extracellular environment is not metabolically inert, rather that it contains various

*Auhor to whom correspondence should be addressed. Abbreviations: PK--protein kinase; PKI--heat and acid

stable protein kinase inhibitor of cAMP-dependent protein kinase; ATP--adenosine triphosphate; cAMP--cyclic adeno- sine monophosphate; HEPES--N-Z-hydroxethyl piperazine-- N'-2-ethane sulphonic acid; HBS--HEPES-buffered saline; BSA--bovine serum albumin; PDBu--phorbol-12,13-dibuty- rate; PKC--protein kinase-C; PKA--protein kinase-A; NCS--new born calf serum; FDA--fluorescein diacetate; PI--propidium iodide; TCA--trichloroacetic acid; CSF-1-- colony stimulating factor 1; GM-CSF--granulocyte macrophage colony stimulating factor.

ecto- and exoenzymes [3]. Both PK and phos- phatases operating at the cell surface can provide a powerful regulatory machinery for extracellular processes analogous to that provided by protein phosphorylation~lephosphorylation systems oper- ating intracellularly [4]. Conditions necessary for protein phosphorylation exist in the extracellular environment, such as divalent cations; ATP; cAMP; ectophosphatase [3-5] and ectoprotein kinase, whose activity was detected on the outer surface of several types of cultured cells [6-12].

Monocytes are bone marrow-derived leukocytes that infiltrate almost all tissues of the body, where they acquire different properties that allow them to perform a variety of different functions. The cells of the monocyte system are involved in the regula- tion of specific immune response, in non-specific defence against infection and malignancy, in inflammation, in regulation of growth of normal and malignant tissue, and in tissue remodelling [13]. Their responses include the secretion of some 100 substances with biological activity ranging from induction of cell growth to cell death [ 14].

423

424 T. GEBERHIWOT and G. SKOGLUND

Thus, through their abundance, distribution, motility and responsivity, monocytic cells interact with almost every cell of the body and extracellu- lar matrix. For instance, when monocytes are recruited from the vasculature into sites of infec- tion or into inflammatory foci, they migrate through the extravascular space by a process of reversible adherence to the extracellular matrix components [15]. Also, in in vitro study, interac- tion between phagocytes and connective tissue proteins such as fibronectin and laminin were shown to enhance opsonin-mediated phagocytosis [16]. As it is possible that these and other plasma membrane associated activities might be regulated at least in part by surface protein phosphoryla- tion-dephosphorylation, we decided to investigate the expression and regulation of ectokinase on U- 937 cells. The U-937 cell line [17] is a monoblas-

toid human tumour cell line with many character- istics of immature monocytes and capable of dif- ferentiation upon appropriate stimulation. We report in this paper the presence of at least three different protein kinases on the surface of intact U-937 cells and a dramatic increase in activity of two of these kinases following cell differentiation.

MATERIALS AND METHODS

Chemicals

Kemptide, phosvitin, cAMP, PKI, ATP, 8-bro- moadenosine 3':5' cyclic monophosphate, HEPES, BSA, Fura 2-AM, trypsin, trypan blue dye and stau- rosporine were purchased from Sigma (St Louis MO, U.S.A.). PKC-peptide, nutrient mixture F-10 and NCS from GIBCO BRL, U.K. IT _32p] ATP (3000 Ci/mMol) were from Amersham (U.K.) GM-CSF and CSF-1 were obtained from Genzyme (Cambridge, MA, U.S.A.). Chemicals for reaction mixture were obtained from Merck (Germany). All other chemicals used were of analytical grade.

Cell culture and preparation

Human Histocytic Lymphoma cell line (U-937) [17], kindly provided by Professor K. Nilson, was maintained in Ham's F-10 medium supplemented with 10% (v/v) NCS, 100 IU/ml of penicillin and 50 lag/ml of streptomycin kept in humidified air at 37°C with 5% CO2. When indicated, cells were induced to differenti- ate into monocytes in the presence of 0.5 mM 8-bro- moadenosine 3':5' cyclic monophosphate and incubated for 48 h [18-20]. Cell viability was evaluated after cell

exposure to phosphorylation conditions by trypan blue exclusion (0.1% in phosphate-buffered saline) and determination of fluorescent cells after simultaneous staining of about 500,000 cells with 50 lal FDA and 15 gl PI. FDA-Pl-stained cells were examined with a stan- dard fluorescence microscope equipped with epi-illumi- nation (Nikon Labphot, Japan). Viable cells fluoresce bright green, while nonviable cells are bright red [21].

Phosphoo'lation assay

The cells were washed twice with pre-warmed HEPES-buffered saline (HBS) [22]. The buffer con- sisted of 0.1% (w/v) BSA, 10 mM glucose, 25 mM HEPES, 120 mM NaC1, 5 ~tM magnesium hexachlo- ride, 6 mM potassium chloride and 1.2 mM calcium chloride (pH 7.4). Routinely, 500,000 cells were incu- bated at 37°C for 10 rain in polypropylene tubes with a final volume of 250 lal containing either phosvitim kemptide or PKC-peptide at a concentration given in the particular experiment. The reaction was initiated by the addition of 0.05 ml [T-32P] ATP at a specific activity of 10 mCi/ml and a final ATP concentration of 1 laM. For phosphorylation of phosvitin, 1 mg/ml of phosvitin was added and the reaction was terminated by adding 2 ml 10% (v/v) TCA. After two cycles of resolubilization with cold 1 M NaOH and reprecipitation with 10% TCA, the pellet was dissolved in 0.2 M NaOH-1% SDS solution and radioactivity was determined by counting aliquots in a scintillation counter (Beckman, LS 5000 CE, U.S.A.) [6]. Kemptide and PKC-peptide, after their incubation period, were treated with a final concentra- tion of 6% TCA-0.08 M acetic acid, and kept on ice for at least 10 rain and then freed from any precipitate by centrifugation (3000 g for 10 rain). Aliquots of the acid- ified cell supernatants were pipetted onto Whatmann P81 filter paper squares (I x 1 cm) and then washed three times in 30% (v/v) acetic acid glycol containing 1% (v/v) phosphoric acid for a minimum of 10 rain, then once with ethanol and then air-dried. Radioactivity was determined by liquid scintillation counting of the papers in 4.5 1111 scintillation fluid [23].

Cell sur]bce tO,psini;ation

In some cases, 5 x l05 cells were suspended in HBS containing 0.25% (w/v) of trypsin for 10 rain at 37°C to remove trypsin sensitive cell-surface proteins [11, 24]. Following removal of trypsin by repeated washing cells were subjected to radiolabelling via the standard proce- dure. Cells remain > 95% viable after trypsin treatment as determined by dye exclusion.

Determination of intracellular free Ca > concentration

Measurement of calcium was performed as described previously [25, 26]. Briefly, cells were loaded with calcium-sensitive fluorescent dye of Fura-2 and

Ectokinase activity on U-937 cells 425

fluorescence was measured with an RF-500 Lpc fluo- rescence spectrophotometer (Shimadzu Corp., Japan).

Statistical analysis and computing

Results were analysed using Student's t-test. Double reciprocal plots of initial reaction velocities were gener- ated using the "Enzyme" computer program from the National Institutes of Health/Kantonspital Winterthur.

RESULTS

Cytophilicity of exogenous substrates

Intact and viable cells are a pre-requisite for the study of ectokinases and the cytophilicity of three selected exogenous substrates were evalu- ated. Table 1 shows the effect of the three sub- strates on intactness of the plasma membrane at concentrations used for routine phosphorylation, in the presence of both positive and negative con- trols. More than 95% of the cells were viable after exposure to phosvitin, kemptide or PKC-peptide, while the previously demonstrated cytotoxicity of histone [6] was also apparent in U-937 cells.

concentration, an increase in initial velocity of phosphorylations was observed, while at higher concentration phosphorylation levelled off (Fig. 1 a). From double reciprocal plots the apparent K m was determined to be 94 + 24 ~tg/ml (mean _+ S.E.M., n = 3). Substrate dependency with kemp- tide was studied in the absence and in the pres- ence of cAMP (5 ~M). Without cAMP a slight but continuous incorporation of radioactivity in a sat- urable process was observed. In the presence of cAMP, phosphorylation was enhanced 3-4-fold and the incorporation increased with increasing kemptide concentration up to 120 IaM (Fig. l b). Double reciprocal plots revealed an apparent Km of 21 + 1.9 ~M (mean _+ S.E.M., n = 3). The PKC- peptide was also phosphorylated in a dose depen- dent manner (Fig. lc). Saturation was achieved at 10 ~m and the Km was calculated to be 2.1 _+ 0.8 ~tM (mean _+ S.E.M., n = 3). With all three sub- strates we observed a time-dependent incorpora- tion of radioactivity that levelled off after about 20 min (Fig. ld).

Phospho~'lation of exogenous substrates by intact U-937 cells

The capacity of intact U-937 cells to catalyse the transfer of 32p from ATP to phosvitin, kemp-

tide and PKC-peptide was investigated and characterized. With increasing phosvitin

Table I. Effects of kemptide, phosvitin, PKC-peptide and his- tone on intactness of plasma membrane

Trypan blue FDA-PI (mean _+ exclusion (mean --+

Cell treatment S.E.M.) S.E.M.)

Buffer 98.0 -+ 0.4 97.2 _+ 0.5 Phosvitin ( 1 mg/ml) 96.2 - 1.2 95. l _+ 3.8 Kemptide ( 130 JaM) 97.0 _+ 0.6 96.0 _+ 1.6 PKC-peptide (10 JaM) 97.7 _+ 0.2 96.8 + 0.4 Histone (100 Jag/ml) 24.8 _+ 3.5 36.2 _+ 8.4

U-937 cells were treated either by trypan blue dye or stained with FDA-PI as detailed in Materials and Methods. For FDA- P1 staining, one hundred fluorescent cells were counted, scored as viable (green) or non-viable (red), and viability cal- culated as number of green cells/(number of green + number of red cells) × 100. Data reported as the mean percentage viable cells + S.E.M. of duplicate count of four different experiments.

Effects of extracellular ATP on extracellular protein phosphorylation and Ca 2+ mobilization

When cells were incubated with phosvitin or kemptide in the presence of different concentra- tions of ATP, incorporation of phosphate into sub- strates increased with increasing ATP concentra- tion and saturation was reached at 100 ~M ATP (Fig. 2a). The K m values of ATP for phosvitin and kemptide phosphorylation were 10.3 _+ 0.7 and 19.5 _+ 4.3 ~tM (mean _+ S.E.M., n = 3), respec-

tively. Since many cellular responses to extracel- lular ATP are mediated by the action of purinergic receptors [3, 4], we performed additional studies to determine the concentration range of ATP uti- lized by purinergic receptors of U-937 cells, by studying the ability of ATP to trigger changes in intracellular Ca 2+ concentrations [25]. ATP con- centrations as low as 0.5 ~tM could trigger mea- surable changes in [Ca2+]i, while increasing ATP above 300 ~m had limited additional effect (Fig. 2b). The K m value for the external ATP regarding Ca 2+ mobilization was determined from double- reciprocal plots and found to be 2.8 _+ 1.2 laM (mean _+ S.E.M., n = 3).

426 T. G E B E R H I W O T and G. S K O G L U N D

1500 's,,

1200

900

600

300 eL

0

(a)

I I I I

400 800 1200 1600 000 Phosvit in conc, (ram)

4OO o

300

% ,~ 20O

100 e~

-6 0

(b)

f ~ p o L-'- - - Z - . . . . . . . . . . . . . . . . . . . . . I x :

I L 100 200

Kemptide conc. (gin) 300

400

300

200

100

o 0

(c)

I

f I I I I

5 10 15 20 25 PKC-peptide conc. (gm) (d)

1500 o

1000

5oo

e~ "6 E 0

.r' 1 I " - - - ' - - ' - - ]

6 12 18 24 30 Time flnin)

Fig. 1. Phosphory la t ion of exogenous proteins by intact U-937 cells. Substrate dependency (a, b and c): 5 × 105 cells in HBS buffer conta in ing 1 lain [./32p] ATP were incubated at 37°C for 10 min in the presence of var ious amounts of phosvit in (a), kemptide either in the presence ( - - ) or absence of 5 ~tM c A M P ( - - ) (b) and PKC-pept ide (c). Radioact ivi ty incorporated into substrates was measured as detailed in Materials and Methods. Time course of radioact ivi ty incorporat ion (d): either 1 mg/ml phosvit in ( - - -) or 125 g M kempt ide plus 5 g M c A M P ( - - • - - ) or 10 g M PKC-pept ide ( ) were treated as above, except the reac- tion was terminated at the indicated times. The results shown are representat ive of three or four separate assays and the mean + S.E.M. of triplicate samples.

Effects qf activators, inhibitors and proteases on ectokinase activiO, of U-937 cells

Table 2 summarizes the effect of different stimulatory and inhibitory conditions for protein kinases on the ectokinase activity. Cyclic AMP and PDBu had no effect on phosvitin phos- phorylation (P > 0.05). Kemptide phosphorylation was stimulated both by cAMP (P < 0.001) and PDBu (P < 0.05). PKC-peptide phosphorylation increased in the presence of either cAMP or PDBu (P < 0.05). Staurosporine and H-7 had no signifi-

cant effect on phosvitin kinase but completely abolished phosphorylation of both kemptide and PKC-peptide (P < 0.001). PKI inhibits cAMP dependency of kemptide phosphorylation, but not PKC-peptide phosphorylation. With the exception of the phosvitin, absence of Mg 2+ from the reac- tion mixture abolished phosphorylation activity. When cells were incubated with 0.25% of trypsin before radiolabelling, phosvitin phosphorylation was markedly decreased (P < 0.05).

Monocytes and macrophages undergo dramatic changes in their phenotype and functional activi-

Ectokinase activity on U-937 cells 427

15,

9

e ,

E 3 -

0 o (a)

~.°

I I [ I 100 200 300 400 500

ATP conc. (.am)

,.~ 3000 ]

o 2 o o o [ / . / ' - - '

1000.

- 0 I I I I 0 200 400 600 800 1000

(b) ATP conc. (gin)

Fig. 2. Response of intact U-937 cells as a function of ATP concentration. Incorporation of phosphate at various ATP concentrations: (a) 5 x l0 s cells containing either l mg/ml phosvitin ( ) or 125 gM kemptide and 5 gM cAMP ( . . . . ) were incubated at 37°C with 0.05 gM [y-~:P] ATP at various ATP concentrations. Radioactivity incorporation into acid-insoluble fractions was determined as detailed in Materi- als and Methods. Results are representative of three separate assays and are the mean -+ S.E.M. of triplicate samples. [Ca2+]~ changes in U-937 cells with various concentration of ATP (b): Cells (1 x I(P) were loaded with Fura-2 as described in Materials and Methods. Cells were washed and re-suspended in a cuvette in a Ca-~+-containing buffer. After determination of baseline Ca :+ concentration, ATP was added at different concentrations and cytosolic [Ca >] in Fura 2-loaded samples were measured at 37°C with continuous stirring. The peak changes in cytosolic [Ca>l were calculated from ATP- induced transients and plotted as a function of extracellular ATP concentration. These Ca -,+ transients were recorded from the same preparations of cells but are representative of the results obtained with four separate preparations of Fura 2- loaded cells.

ties when activated [18, 20]. We thus tested the effect of some monocyte activating substances on the ectokinase activity of U-937 cells. However, short term treatment with CSF-1, GM-CSF and leukotriene C4 had negligible effect on the phos- phorylation of all substrates (P > 0.05).

Table 2. Effects of activators, stimulators or inhibitors on the phosphorylation patterns

Agents Phosvitin Kemptide* PKC-peptide

Control 2140 -+ 140 193 _+ 75 458 -+ 6.30

CSF 2217__220 588+-9.8 479-+ 12.5

GM-CSF 2140± 106 582 ± 11 471 + 10.8

LTC4 2153 +- 256 582 -+ 8.5 465 + 15.5

Cyclic AMP 2063 +- 144 580 _+ 8.7 553 +- 55.0

PDBu 2115_+77 316+-3.6 672+-10.6

Staurosporine 1809 +- 53 3.3 +- 2.1 5.7 -+ 2.30

H-7 1837-_+ 182 10_+4 4.9+- 1.60

Mg-exclusion 1720 ± 264 I 1 _+ 1.2 5.7 +- 3.20

PKI - - 182 ± 5.2 437 ± 46.0

Trypsin 726 -+ 32 - - - -

U-937 cells (5 x 10 ~) and substrates were pre-incubated for 5 rain in the presence of the indicated agents. Reaction was started by adding [7-32P] ATP in the pre-incubated reaction mixture and phosphorylation was determined as described in Materials and Methods. CSF-1 (50 IU), GM-CSF (20 ng/ml), LTC4 (10 nM), PDBu (200 nM), cAMP (5 gM), staurosporine (100 nM), (H-7 10 IJM), PKI (1500 gg/ml), phosvitin (1 mg/Ml), kemptide (130 p.M), PKC-peptide (10 btM) and trypsin (0.25% w/v) were used. Results are reported as phos- phate incorporated counted per minute and are the mean -.- S.E.M. of three different experiments assayed in triplicate.

*Except for the control and PDBu stimulated, all kemptide phosphorylations were performed in the presence of 5 laM cAMP.

7000

~---~ 6000 ~ None-differ.

I I Differ. 5ooo

o4000 Ea

3OOO ._=

2ooo ........ i~

o I000 N " "

0 Phnsvitin Kemptide PKC-pep

Fig. 3. Effects of U-937 cell activation on phosphorylation pattern of the three substrates. In addition to the routine cul- ture medium, 0.5 mM 8-bromoadenosine cyclic monophos- phate was included in the medium and incubated for at least 48 h. Control groups of cells were propagated in the absence of cyclic nucleotide. Cells 5 x 106 were subjected to phospho- rylation conditions in the presence of 1 mg/ml phosvitin, 125 laM kemptide and 5 pM cAMP, or 10 gM PKC-peptide as detailed in Materials and Methods. Results are reported as phosphate incorporated counted per minute. Data are the mean + S.E.M. of three different experiments assayed in trip- licate.

428 T. GEBERHIWOT and G. SKOGLUND

Effects of cell differentiation on extracellular kinase activi~

Figure 3 shows that cells differentiated for 48 h in the presence of 0.5 mM 8-bromoadenosine 3':5' cyclic monophosphate have more than double phosphorylating capacity for phosvitin and PKC- peptide compared with non-differentiated (P < 0.001), whereas kemptide phosphorylation was unchanged by cell differentiation (P > 0.05).

DISCUSSION

In this study, we demonstrated the existence of at least three ectoprotein kinases on the surface of U-937 cells. To the study of reactions restricted to the outer aspect of the cell, the intactness of the cell is of crucial importance. Participation of intracellular protein kinases during the reaction was improbable for the following reasons: (1) no cell lysis was demonstrated either by trypan blue exclusion or FDA-PI staining [21]; (2) incubation of cells with [7-32p] ATP, which does not pene- trate cells [3, 4], resulted in radiolabelling of the reaction products in the extracellular environment; (3) an absolute requirement of extracellular Mg :+ /'or most phosphorylation supports an extracellular reaction; (4) an increase in phosphorylation activ- ity was detected when cells were lysed (results not shown); (5) addition to intact cells of PKI, which does not cross the membrane bah'let within the limited time, inhibits cAMP-dependent kemp- tide phosphorylation; (6) proteolysis of the ectoki- nase from intact U-937 cells by 0.25% of trypsin also suggests that these proteins are located on the cell surface. These characteristics are in line with those of other studies done on extracellular pro- tein phosphorylation [6-11 ].

To characterize the protein kinases, we have selected three different substrates which showed a degree of specificity for particular protein kinases. Firstly, phosvitin phosphorylation (casein kinase II) is lbund to be independent of either cAMP or PDBu, suggesting that the reaction involves nei- ther PK-A nor PK-C. This is in agreement with previous studies [4, 6]). Secondly, as reported pre- viously for HeLa cells [8], we have shown that kemptide phosphorylation in the presence of

cAMP increased 3- to 4-fold and its cAMP depen- dency was abolished in the presence of heat- and acid-stable specific inhibitory protein for cAMP- dependent protein kinase. It is particularly impor- tant to note, however, that the basal phosphoryla- t/on is unchanged by PKI alone, suggesting the additional existence of cAMP-independent ecto- PK phosphorylating kemptide. This is further sup- ported by our finding that PDBu, instead of cAMP, as a regulatory agent increased kemptide phosphorylation and that H-7 and staurosporine also abolish the basal phosphorylation of kemp- tide. Thirdly, PKC-peptide phosphorylation was partially stimulated with PDBu and cAMP, and inhibited completely by H-7 and stau- rosporine. Unlike kemptide, PKI had no signifi- cant effect on PKC-peptide phosphorylation. In addition to the above mentioned difference, dur- ing cell differentiation PKC-peptide phosphoryla- t/on as well as phosvitin phosphorylation was doubled, whereas kemptide phosvitinphosphoryla- t/on was unchanged. It is also notable that phos- phorylation does not seem to require the addition of Mg >, thus marking further distinction from the other two activities. This partial Mg 2+ indepen- dence of extracellular phosvitin phosphorylation is comparable to a previous study of neuronal cells 1271. Taken together, the collected evidence sug- gests that U-937 cells on their surface possess phosvitin (casein) kinase, cAMP-dependent kinase and a kinase capable of phosphorylating a PKC- peptide in a phorbol ester stimulatable manner.

The apparent Kin-values for ATP (10 20 mM) regarding phosphorylation of kemptide and phosvitin are comparable to the K,,,-values reported /'or intracellular kinases [1]. Our finding that ATP-stimulated (purinergic receptor-medi- ated event) increase in intracellular Ca -'+ [25] comes into operation in the same ATP concentra- tion range, gives additional support to the idea that ectokinases can and will operate under physi- ological conditions.

When U-937 cells are treated/br 48 h with 0.5 mM 8-bromoadenosine cyclic monophosphate, growth is arrested; cells differentiate morphologi- cally and functionally, exhibiting an increase in many properties characteristic of macrophages

Ectokinase activity on U-937 cells 429

[20-23]. In our study, cell differentiation signifi- cantly increased phosvitin and PKC-pept ide phos- phorylation, a finding comparable to a previous study of neuronal cells [7]. Unlike the other two substrates, kemptide phosphorylation was unchanged. The finding that increased phosphory- lation of cell surface proteins (unpublished data), phosvitin and PKC-pept ide by ectoprotein kinases following cell differentiation indicates that the extracellular phosphorylation system is associated with some specialized functions of macrophages.

In conclusion, the U-937 cell line appears to possess a further means for modulation of extra- cellular proteins by different ectokinase activities. This may constitute another regulatory modali ty for the interaction of monocytic cells with the extracellular environment in cel l -cel l contact, ce l l -matr ix contact, cell motility and signal trans- duction as well as growth and cell differentiation. The differential changes observed in phosphoryla- tion capacity of different kinases at different mat- urational stages may be valuable in the study of the physiological role of ectoprotein kinase activ- ity, which is still a matter of speculation.

Work is in progress to characterize the natural substrates of these kinases both in serum and on the cell surface, and the significance of their phos- phorylation.

Acknowledgements~This work was conducted as part of collaboration between Addis Ababa University, Faculty of Medicine and Karolinska Institutet and was supported by SAREC (Swedish Agency for Research Co-operation with Developing Countries) (grant No. S2/ETI 12).

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