Life Sciences, Vol. 48, pp. 995-1005 Printed in the U.S.A.

PROTEIN

Pergamon Press

P H O S P H O R Y L A T I O N SUBSTRATES IN N O R M A L AND N E O P L A S T I C OF THE H U M A N U P P E R AERO-DIGESTIVE TRACT.

Ewa L. Rydell I, Jan Olofsson4~

SSlve Hellem 2 and

SQUAMOUS

EPITHELIA

Krister L. Axelsson 1'3

Departments of Pharmacology I and Oral Surgery 2, Faculty of Health Sciences, Department of Biology 3, University of LinkSping, S-581 85 LinkSping, Sweden and Department of Otolaryngology/Head & Neck Surgery 4, University of Bergen, Norway. (Received in final form January 7, 1991) SUMMARY Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriehed preparations from squamous cell carcinomas and normal mueosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 5255 kDa. Addition of Ca2+or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca 2+ /phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heatstable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors. Protein phosphorylation is an essential element in the transduction of certain cellular signals. In addition, protein phosphorylation is involved in the regulation of cell growth and differentiation (for review see 1,2). However, there is still much to be discovered regarding the exact mechanisms through which these effects are exerted. Phosphorylation is mediated by several different protein kinases. One of these, protein kinase C, has been 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc

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extensively studied since its discovery in the late seventies. Several proteins, such as epidermal growth factor receptor, myosin light chain and gap junction proteins, serve as phosphorylation substrates for protein kinase C both in v i t r o and in v i v o (3-5). Furthermore, a protein phosphorylation substrate for Pk-C with reported molecular weight between 80-90 kDa has been found in several different cells (6). In a previous study, using exogenous substrates, we have shown an increased activity of Pk-C in squamous cell carcinomas when compared to the Pk-C activity found in normal mucosa (7). Furthermore, we found an increased basal protein kinase activity in these tumors. In the present study we have attempted to further clarify the basis for the increased basal protein kinase activity, and to investigate the existence of endogenous phosphorylation substrates in squamous cell carcinomas and normal mucosa from the human upper aero-digestive tract. MATERIALS AND METHODS Preparation of cellular extracts Extracts were prepared from squamous cell carcinomas or normal mucosa from the upper aero-digestive tract. Tumors from i0 patients ranging in age between 46-65 years were used. Normal mucosa from 12 patients undergoing surgery for non-neoplastic disease were used as control. Oral mucosa obtained in the present study contained about 70-80 % epithelial tissue. The specimens were placed in ice-cold 0.9 % NaCI and frozen at -70 °C within 10-15 min following their surgical removal. Extracts were prepared by homogenizing in 20 mM TrisHCI pH 7.5 containing 2 mM EDTA, I m_M EGTA and 50 mM mercaptoethanol in a polytron for 3 x I0 sec. Homogenates were centrifugated at 40,000 x g for 25 min. In some experiments, the protease inhibitor leupeptin (30 Bg/ml) was included in the homogenization buffer. Supernatants were collected and used as crude soluble sources of enzyme. Pellets were resuspended in homogenization buffer together with 0.I % Triton X-100 and left on ice for 1 h. Subsequent centrifugation as described above resulted in the solubilized particulate fraction. Preparation of the tissue extracts was carried out at 4 °C. Partial purification of soluble Pk-C was performed by anion-exchange chromatography on a DE-52 column (3.5 x 0.75 cm), equilibrated with homogenization buffer (8). After application of the sample (3 mg of protein), the column was washed with homogenization buffer. Pk-C was step-wise eluted with 50-400 mM NaCl in homogenization buffer. Fractions of 1 ml was collected. Pk-C eluted at 100-150 mM NaCI, the peak fraction containing 85-90 % of the total Pk-C activity. This Pk-C-enriched fraction was used for endogenous phosphorylation assays. In vitro phosphorylation

assay and polyacrylamide

~el electrophoresis.

The total assay volume was 200 ~i and the assay medium had the following composition: Tris-HCl (50 m_M, pH 7.5), MgCI 2 (I0 mM), phosphatidylserine [PS] (20 ~g), 1,2-diacylglycerol [DAG] (I ~g), CaCI 2 (0.7 mM free concentration) and ATP (5 ~M) containing [y-32p]ATP (0.8-1.3 x 107cpm) and extract from the crude soluble or particulate fraction (20-85 ~g protein) or from the Pk-Cenriched fraction (10-20 ~g protein). Basal phosphor~lation was determined in the absence of phospholipids and exogenously added Ca ~+ but in the presence of 4 mM EGTA (to minimize any effect of endogenous Ca2+). The different inhibitors, when used, were present in the following concentrations: 1.3 ~M (Wiptide), 1 ~g/ml (heat-stable Pk-A inhibitor), 22.3 ~H (Pk-C inhibitor peptide 19-36), I00 ~M ~H-7) and 2 ~g/ml (heparin). The reaction was initiated by the addition of [y-~ZP]ATP, run for 5 min (unless otherwise indicated) at 30 °C and stopped by the addition of 100 ~i stop solution (0.1875 M Tris-HCl pH 8.8, 6 % SDS, 30 % glycerol, 15 % mercaptoethanol). The reaction mixtures

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were boiled for 3 min. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the p h o s p h o r y l a t e d samples was carried out in 15 % separating gels according to the method of Laemmli (9). In some experiments, I0 % separating gels were used in order to allow resolution of proteins with m o l e c u l a r weights above 100 kDa. The gels were dried and exposed to Kodak X-OMAT AR film at -70 °C. Reactions run in parallel were stopped by the addition of 2 ml 5 % TCA and 200 ~i 0.3 % bovine serum albumin (BSA). An additional 2 ml 5 % TCA was added. The resultant precipitates were centrifuged at 1,600 x g for 2 min, supernarants were removed and precipitates dissolved in 200 ~I 0.2 M NaOH. Protein was re-precipitated by addition of 2 ml 5 % TCA, centrifuged and resolubilized. This washing procedure was conducted 3 times. Finally, 5 ml of toluene-based scintillation cocktail was added to each alkali-dissolved precipitate and the samples were counted in a LKB Wallac liquid scintillation counter. Protein

determination

and statistical

evaluation.

Protein was determined according to the method of Bradford (i0) using a commercial protein assay reagent (Pierce Chemical Company, Iii, U.S.A.), and bovine serum albumin (BSA) as standard. Results are given as means ± SEM and are expressed as pmole incorporated 32p/mg protein/min. Statistical significances were calculated w i t h Student's t-test on unpaired observations. The effect of the various inhibitors was calculated with Wilcoxon signed-rank test on ralative changes in enzyme activity. Net endogenous Pk-C-dependent phosphorylation was calculated by subtracting the activity in the presence of Ca 2+ from the activity in the presence of Ca 2+ and phospholipids. Chemicals. Molecular weight standards were purchased from Pharmacia AB, Uppsala, Sweden. Electrophoretic reagents were purchased from Bio-Rad Laboratories, Richmond, Ca, U.S.A. Peptide 19-36 and Pk-A inhibitor peptide (Wiptide) were from Peninsula laboratories, Belmont, CA, U.S.A. Other chemicals were from Sigma Chemicals Co., St. Louis, Mo., U.S.A. (y- 32 P)-ATP was purchased from NEN Chemicals, Dreiech, FRG. RESULTS Basal endogenous phosphorylation was about 5-fold higher in crude soluble fractions from squamous cell carcinomas compared to extracts from normal mucosa. In particulate fractions the basal endogenous phosphorylation was 3fold higher in tumors when compared to normal mucosa (Tab. 1 and Fig. I). Addition of 1 mM Ca 2+ alone or I mM Ca 2+ in the presence of PS/DAG had only a slight effect on the endogenous phosphorylation in crude extracts from either tissue (Tab. I). Furthermore, neither cyclic AMP (0.5 ~M) nor cyclic GMP (0.5 ~M) had any effect on the endogenous protein phosphorylation (data not shown). When the protease inhibitor leupeptin (30 ~g/ml) was included in the reaction mixture there was no difference in basal protein p h o s p h o r y l a t i o n over the time range 0-20 min (data not shown). The Pk-C dependent protein phosphorylation was higher in the presence of leupeptin and showed linearity over the time range of 2-5 min both in the presence and absence of leupeptin. However, after 5 min the phosphorylation levelled off (Fig. 2). Studies revealed

of a

autoradio~rams obtained from soluble number of 3~p-labelled protein bands,

extracts run in SDS-PAGE among them a band in the

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r e g i o n of 45-50 kDa w h i c h was only o b s e r v e d in e x t r a c t s f r o m t u m o r s p h o s p h o r y lated in the p r e s e n c e of Ca 2+ or Ca 2+ and P S / D A G (Fig I., lane 5 and 6). O t h e r phosphorylated proteins w h i c h w e r e only d e t e c t e d in soluble extracts from t u m o r s had a p p a r e n t m o l e c u l a r w e i g h t s of 18, 33, 37, 52, 60, 62 and 90 kDa (Fig. i). Several bands c o r r e s p o n d i n g to p r o t e i n s w i t h a p p a r e n t molecular w e i g h t s of 38, 41, 45, 51, 56 and 76-80 kDa were seen in crude p a r t i c u l a t e fractions of tumors. In a d d i t i o n three p h o s p h o r y l a t e d p r o t e i n bands with apparent m o l e c u l a r w e i g h t s of about 20, 28 and 63 kD were o b s e r v e d specific a l l y in the p r e s e n c e of Ca 2+ and p h o s p h o l i p i d s .

TABLE

I.

Endogenous phosphorylation under basal c o n d i t i o n s and in the p r e s e n c e of 0.7 mM Ca 2+, 0.7 mM C a 2 + / p h o s p h a t i d y l s e r i n e (PS)/I,2diacylglycerol (DAG), and net p r o t e i n k i n a s e C (Pk-C) p h o s p h o r y l a tion in crude soluble and p a r t i c u l a t e extracts, and in Pk-Ce n r i c h e d f r a c t i o n s o b t a i n e d as d e s c r i b e d in m a t e r i a l s and methods. Net e n d o g e n o u s Pk-C d e p e n d e n t p h o s p h o r y l a t i o n was c a l c u l a t e d by subtracting the p h o s p h o r y l a t i o n in the p r e s e n c e of Ca 2+ from the that in the p r e s e n c e of Ca 2+ and phospholipids. [pmol 32p/mg prot/min. M e a n + SEM; n=5-7].

Normal mucosa Crude soluble extract

Crude particulate extract

Ca2+

3.3 + 0.8

3.4 + 1.0

Ca2+/ PS/DAG

5.0

+ 2.2

(p = 0.001) Tumor

Normal mucosa Soluble Pk-C-enriched extract

Basal

Net

Pk-C

1.2 _+ 0.8 (p > 0.I)

14.7 + 2.6

14.9 _+ 2.4

13.9 + 2.9

2.5 + 1.7

2.3 + 0.i

2.1 + 0.3

7.8 + 1.6

5.6 _+ 1.4

(p < 0.02)

(p < 0.05)

Tumor

6.7 + 1.3

9.1

+ 2.0

28.7

+ 7.0

19.6 + 5.8

Normal mucosa

9.8 + 2.9

6.2 + 0.I

I0.0

+ 1.5

3.8 ± 1.4

(p < 0.01) Tumor

25.5

_+ 2.1

(p < 0.05) 27.4

_+ 6.5

26.0

_+ 5.8

0.0 + 0.0

In p a r t i c u l a t e e x t r a c t s of normal tissue the 51, 56 and 76-80 kD bands were most pronounced, and no effect of could be o b s e r v e d (Fig. 3), SDS-PAGE in I0 % s e p a r a t i n g gels, which resolves proteins with molecular w e i g h t s above I00 kDa, revealed one m a j o r p h o s p h o r y l a t e d p r o t e i n w i t h m o l e c u far w e i g h t of 120-130 kDa in the p r e s e n c e of Ca 2+ and Ca 2+ /PS/DAG. This was seen in both soluble and p a r t i c u l a t e fractions of tumors. Addition of l e u p e p t i n (30 ~g/ml) to the r e a c t i o n m i x t u r e had no effect on the p h o s p h o r y l a tion p a t t e r n (data not shown).

Ca2+/pS/DAG

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In order to determine whether the apparent elevated basal phosphorylation in tumor extracts was due to an endogenous activation of protein kinases, the effects of different protein kinase inhibitors were investigated. These inhibitors included: heat-stable cAMP-dependent protein kinase (Pk-A) inhibitor (I ~g/ml), Pk-A inhibitor peptide (1.3 ~M) (II), heparin (2.0 ~g/ml) as a putative inhibitor of casein kinase II (12), and the two PK-C inhibitors H-7 (I00 ~M) and peptide 19-36 (22.3 ~M) (13, 14). None of the inhibitors had any significant effect on the basal endogenous phosphorylation in neither soluble nor particulate extracts from tumors or normal mucosa (Table 2).

Lane

1

2

3

4

5

6 -94 -67 - 43

- 30

- 20.1 Fig.

I.

Autoradiogram showing endogenous protein phosphorylation pattern in crude soluble extract from normal mucosa (lane I-3) and tumors (lane 4-6). Lane 1 and 4; basal activity, lane 2 and 5; Ca2+(0.7 mM) and lane 3 and 6: Ca2+(0.7 mM) and PS/DAG. 5 ~g of protein was added to each lane. The positions of molecular weight markers (in kDa) are shown on the right. Representative gel from 8 different experiments. The film was exposed for 7 days. In an attempt to further characterize Pk-C-dependent protein phosphorylations, fractions enriched in Fk-C activity were obtained by anion-exchange chromatography of crude soluble extracts. These fractions contained proteins which were phosphorylated under basal conditions, and the difference in basal endogenous phosphorylation between tumors and normal mucosa was also evident in this fraction (Tab. i). Calciurn(l mM) in the presence of PS/DAG caused a 4-5 fold stimulation of endogenous protein phosphorylation (Tab. I). The difference between tumors and normal mucosa with regard to Pk-C-mediated phosphorylation of endogenous proteins is also evident from autoradiograms obtained after SDS-PAGE (Fig. 4). The p h o s p h o r y l a t i o n intensity was much more pronounced in the Pk-C enriched extracts from tumors compared to extracts from normal mucosa, and three bands

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200'

O----

¢q

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i

loo

o'3

0

10

20

Time (rain) Fi 8 . 2 Protein kinase C dependent phosphorylation in crude soluble extracts of tumors in the absence (-) and presence (+) of 30 ug/ml leupeptin. Mean values of 3 experiments.

NORMAL

TUMOR

i

-94

I

--67

i -43

-30

-20.1 -14.4

O

O

m

O

O

Fi B . 3. Autoradiogram showing endogenous protein phosphorylation pattern in crude particulate extract from normal mucosa and tumors. The position of molecular weight markers (in kDa) are shown on the right. 8 Bg of protein was added to each lane. Representative gel from 3 different experiments. The film was exposed for i0 days.

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TABLE 2 Effect of different protein kinase inhibitors on basal endogenous protein phosphorylation in crude soluble and particulate tissue extracts from tumors. Results are expressed as percentage of protein phosphorylation in the absence of inhibitors (control). Mean ± SEM (n=3-5).(ND=not determined) Treatment

Protein kinase activity of control. soluble

Control

particulate

I00

Pk-A inhibitor

(I pE/ml)

in %

i00

III.0 + 6.3

94.7 + 12.6

Wiptide

(1.3 pM)

110.9 _+ 3.7

91.0 + I0.0

Heparin

(2 Bg/ml)

82.3 + 9.4

ND

Peptide

19-36 (22.3 ~M)

109.5 + 3.0

83.5 _+ 13.3

l-(5-Isoquinolinylsulfonyl)2-methylpiperazine [H-7]

86.3 + 10.3

ND

(ioo ~M)

Lane

1

2

3

4

5

6 - 94 -67

- 43

- 30 -20.1 Fi 8 . 4. A u t o r a d i o g r a m showin 8 endogenous protein phosphorylation patterns in the Pk-C-enriched fractions obtained as described in materials an methods from crude tissue extracts. Tumors (lane 1-3) and normal mucosa (lane 4-6). Conditions are the same as in fig. 2. 4 Mg of protein was added to each lane. Representative Eel from 5 different experiments. The film was exposed for 5 days.

1001

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I

43

30

I

I

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20.1 I

Fig. 5. A u t o r a d i o g r a m showing the effect of the protein kinase C inhibitor peptide 19-36 on the Ca2+/PS/DAG-stimulated endogenous protein phosphorylation in the Pk-C-enriched fraction from tumors obtained as described in materials and methods. Lower lane: Control. Upper lane: + peptide 19-36 (22.3 ~M). The position of molecular weight markers (in kDa) are indicated on top. Representative gel from 3 different experiments. 5 ~g of protein was added to each lane. The film was exposed for 5 days. corresponding to proteins with apparent molecular weight of 18, 30 and 48 kDa were detected exclusively in extracts of tumor tissue. The Pk-C mediated phosphorylation was almost completely inhibited by inclusion of a specific PkC inhibitor (peptide 19-36) in the incubation medium (Fig. 5). DISCUSSION In the present study we have demonstrated a significantly elevated basal phosphorylation of endogenous proteins, as well as an increased Pk-C-dependent p h o s p h o r y l a t i o n of endogenous proteins in neoplastic squamous epithelia from the human upper aero-digestive tract compared to normal epithelia. These results support our previous findings, which showed a marked increase in basal phosphorylation as well as Pk-C mediated phosphorylation of exogenously added histones in tissue extracts from tumors compared to normal mucosa (7). We have also shown that casein kinase II activities are elevated in soluble extracts of tumors compared to normal mucosa (15). The cause of the elevated basal phosphorylation could be due to endogenous activation of protein kinases. Therefore, the effects of different inhibitors of serine/threonine protein kinases (Pk-C, Pk-A and casein kinase II) were investigated. None of the inhibitors had any significant effect on the basal endogenous phosphorylation. These results seem to argue against the possibility that increased activities of the above mentioned protein kinases explain the higher basal protein phosphorylation in extracts from tumors. Several other serine/threonine protein kinases are known such as cyclic GMP dependent protein kinase (Pk-G), and an increased activity of these could contribute to the higher basal phosphorylation in tumor tissue (16). Studies of partially purified Pk-A and Pk-G could most probably contribute to the explanation of the higher basal phosphorylation activity in tumors when compared with normal tissue. Furthermore, tyrosine kinases may also be of importance. Studies concerning tyrosine protein kinases are at present in progress in our laboratory. Phosphorylation of endogenous proteins in crude soluble tissue extracts from tumors with subsequent SDS-PAGE and autoradiography, consistently revealed several bands which corresponded to phosphorylated proteins. In normal mucosa

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the phosphorylation of these proteins was very low or absent. Addition of Ca 2+ either alone or in the presence of PS/DAG induced the phosphorylation of additional proteins in tumor extracts but not in extracts from normal mucosa. The poor effect of Ca/PS/DAG on the phosphorylation in crude extracts as shown in Tab. I can have several explanations: i. Absence of Pk-C in this tissue; 2. Maximal activation of Pk-C in the tissue extract even prior to addition of Ca/PS/DAG; 3. Absence of endogenous substrates for Pk-C; 4. Masking of the Pk-C phosphorylating activity by the high basal phosphorylating activity; 5. High amount of catalytic fragments of Pk--C; 6. The presence of endogenous protein kinase inhibitors. The first two alternatives can be ruled out since we have previously shown Ca/PS/DAG-stimulated phosphorylation in the presence of exogenous histones in epithelial tissue (7). Furthermore, after purification of tissue extracts from tumors and normal mucosa by anionexchange chromatography, Pk-C specific phosphorylation of endogenous proteins could be detected in the peak fraction enriched in Pk-C, indicating that Pk-C phosphorylating substrates are present in these tissues. It should be emphasized that t h e s e phosphorylation substrates which coelute with Pk-C most likely are not the only Pk-C substrates present in the tissue. They can also be substrates for other protein kinases. Catalytic fragments of Pk-C obtained after Ca2+-dependent protease cleavage loose their requirement for Ca 2+ and phospholipids and are fully active even in the absence of these activators (I, 17). High amount of these fragments can contribute to the elevated basal protein phosphorylation and the lack of Ca 2+ /PS/DAG dependency. However, after partial purification of crude soluble extracts a pronounced Ca 2+ and phospholipid-dependent protein kinase activity was seen and therefore the crude extract must contain relatively high amounts of intact Pk-C. The presence of endogenous protein kinase C inhibitors in various tissues has been demonstrated (18, 19), and the presence of such inhibitors might explain the poor Ca/PS/DAG-dependency. It is possible that these inhibitors are removed during anion exchange chromatography of tissue extracts which thus would explain the marked effect of Ca2+/PS/DAG in the Pk-C enriched fractions. .... Three proteins with apparent molecular weights of 18, 30 and 48 kDa were consistently seen in the Pk-C-enriched fractions of extracts from tumors but not in normal tissue. The phosphorylation of these proteins was almost completely inhibited in the presence of Pk-C inhibitor peptide, substantiating the assumption that these proteins are substrates for Pk-C. The differences in protein phosphorylation activity between normal and tumor tissue extracts, thus seem to be due to alterations in both the occurrence and abundance of phosphorylation substrates, as well as in PK-C activity. Differences in phosphatase activities can also be of importance. In this case, lower phosphatase activities would be seen in tumor extracts when compared to normal mucosa. Preliminary results from our laboratory do not, however, support this theory, since we have found about 3-fold higher activities of both alkaline and acid phosphatases in tumors compared to normal tissue (unpublished observations). Numerous endogenous proteins, such as epidermal growth factor receptor, gap junction proteins and protein components to the cellular contractile apparatus, have been found to be phosphorylated by both cytosolic and membranebound Pk-C in vitro (3, 5, 20). The identity of the phosphorylated proteins in the present study are unknown. The proteins of 18-20 kDa in soluble and particulate extracts as observed in our study have previously been reported in experimentally induced rat thyroid goiters, rat adrenal glomerulosa cells and human neutrophils (21-23). The 90 kDa band could correspond to the 87 kDa protein seen in isolated nerve terminals and in the cytosol of brain, spinal cord and heart (6, 24-27). A 80-87 kDa alanine-rich substrate has been cloned and characterized, and mRNA for this substrate is highly expressed in brain

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and other tissues (28). The protein with apparent molecular weight of about 30 kDa seen in our study might correspond to the observed 27 kDa gap junction protein phosphorylated in v i t r o by Fk-C (5). This is interesting, since the gap junction channels provide an important pathway for communication between cells, and phorbol esters, direct activators of protein kinase C, have been found to inhibit cell-to-cell communication (29, 30). ACKNOWLEDGEMENT This study was supported by grants from the Swedish Society for Medical Research, Ingrid Svenssons foundation for cancer research, The Swedish Medical Research Council (08292), and the Norwegian Cancer Society. REFERENCES I. Y. NISHIZUKA, Science. 233 305-312 (1986) 2. R.C. SCHATZMANN, R.S. TURNER and J.F.KUO, Calcium and Cell function, Wai Yiu Cheung (ed), Vol. V 33-66 Academic Press, New York (1984) 3. T. HUNTER, N. LING and J.A. COOPER, Nature 311 480-483 (1984) 4. T. END0, M. NAKA and H. HIDAKA, Biochem. Biophys. Res. Commun. 105 ~. 942-948 (1982) 5. A. TAKEDA, E. HASHIMOTO, H. YAMAMURA and T. SHIMAZU, FEBS Lett. 210 "169-172 (1987) 6. K.A. ALBERT, S.I. WALAAS, J. K-T. WANG and P. GREENGARD, Proc. Natl. Acad. Sci. USA. 83 2822-2826 (1986) 7. E.L. RYDELL, K.L. AXELSSON and J. OLOFSSON, Sec. Mess. Phosphoprot. 12 155-162 (1988) 8. R.KUMAR and O.HOLIAN, J. Invest Dermatol. 86 316-320 (1986) 9. U.K. LAEMMLI, Nature 227 680-685 (1970) I0. M.M. BRADFORD, Anal. Biochem. 72 248-254 (1976) II. H.C. CHENG, B.E. KEMP, R.B. PEARSON, A.J. SMITH, L. MISCONI, S.M. VAN FATTEN and D.A. WALSH, J.Biol. Chem. 261 989-992 (1986) 12. K.-P. HUANG, E. ITARTE, T.J. SINGH and A. AKATSURA, J. Biol. Chem. 257 3236-3242 (1982). 13. H. HIDAKA, M. INAGAKI, S. KAWAMOT0 and Y. SASAKI, Biochemistry. 23 5036-5041 (1984) 14. C. HOUSE and B.E. KEMP, Science. 238 1726-1728 (1987) 15. E. RYDELL, K.L. AXELSSON, J. OLOFSSON and S. HELLEM, Cancer Biochem. Biophys. (1990)(in press) 16. S.K. HANKS, A.M. QUINN and T. HUNTER, Science 241 42-52 (1988) 17. Y. NISHIZUKA Nature 308 693-698 (1984) 18. N. SCHWANTKE and C.J. LE PEUCH, FEBS Letters 177 36-40 (1984) 19. K. M. EYESTER, Biochem. Biophys. Res. Comm. 168 609-615 (1990) 20. T. NAKAKI, B.C. WISE and D.-M, CHUANG, Life Sci. 42 1315-1321 (1988). 21. B. OMRI, M.F. BRETON and M. PAVLOVIC-HOURNAC, Mol. Cell. End. 48 105II0 (1986) 22. T. KIGOSHI, K. USHIDA and S. MORIMOTO, J. Steroid. Biochem. 29 277283 (1988) 23. D.M. HELFMAN, B.D. APPELBAUM, W.R. VOGLER and J.F. KUO, Biochem. Biophys. Res. Comm. iii 847-853 (1983) 24. J.K.T. WANG, S.I. WALAAS, T.S. SIHRA, A. ADEREM and P. GREENGARD, Proc. Natl. Acad. Sci. USA. 86 2253-2256 (1989) 25. W.C.-S. WU, S.I. WALAAS, A.C. NAIRN and P. GREENGARD, Proc. Natl. Acad. Sci. USA. 79 5249-5253 (1982) 26. S.l. WALAAS, A.C. NAIRN and P. GREENGARD, J. Neurosci. 3 291-301 (1983) 27. S.I. WALAAS, A . C . NAIRN a n d P. GREENGARD, J. Neurosci. 3 302-311 (1983) 28. D.J. STUMPO, J.M. GRAFF, K.A. ALBERT and P. GREENGARD, Proc. Natl.

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Acad. Sci. USA. 86 4012-4016 (1989). 29. A.W. MURRAY and D.J. FITZGERALD, Biochem. Biophys. Res. Comm. 91 395401 (1979) 30. H. St. C. GAINER and A.W. MURRAY, Biochem. Biophys. Res. Comm. 126 1109-1113 (1985)