Protein phosphorylation in type II pneumocytes DAVID WARBURTON, LINA COSICO, AND

and dephosphorylation

AGNES TAYAG, RAJEEV SETH

SUE BUCKLEY,

Developmental Lung Cell and Molecular Biology Research Center, Cell Biology Group and Division of Neonatology and Pediatric Pulmonology, Childrens Hospital of Los Angeles, University of Southern California School of Medicine, Los Angeles, California 90027

WARBURTON,

DAVID, AGNES TAYAG, SUE BUCKLEY, RAJEEV SETH. Protein phosphorylation

LINA

and dephosphorylation in type II penumocytes. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L548-L554, 1991.-Protein phosphorylation and dephosphorylation are a major mechanism for regulating cellular activity. Substantial evidence exists for ascribing a key role of protein phosphorylation and dephosphorylation in the regulation of surfactant secretion from type II pneumocytes, yet understanding of the specific molecular mechanisms is generally lacking. Herein, we report two-dimensional electrophoretic mapping of proteins phosphorylated in type II pneumocytes in primary culture, the response to protein kinase C simulation with phorbol ester, and the response to protein phosphatase inhibition with okadaic acid. Exposure of cells for 15 min to phorbol ester at a concentration (10s4 M) which maximally stimulated both translocation of protein kinase C from cytosol to membranes and surfactant secretion increased phosphorylation (50-80% compared with control) of three specific proteins (50 kDa, p1 5.8 and 5.7; 25 kDa, p1 5.7). Exposure of cells for 2.5 h to okadaic acid (10s6 M), a concentration that inhibited 90% of protein phosphatase activity, resulted in greatly increased phosphorylation (200-1,500% compared with control) of five specific proteins (50 kDa, p1 5.7 and 5.6; 45 kDa, ~15.5; 40 kDa, ~15.5; 25 kDa, ~15.5). Combined treatment with okadaic acid and phorbol ester resulted in further increases (145-3,080% compared with control) in phosphorylation of four specific proteins (50 kDa, p1 5.6; 45 kDa, p1 5.5; 40 kDa, p1 5.5; 25 kDa, p1 5.5). We conclude that these respective proteins comprise major substrates for protein kinase C-dependent phosphorylation and for protein phosphatases in type II pneumocytes in primary culture. Furthermore, we speculate that these proteins will prove to play key roles in the regulation of type II pneumocyte function. COSICO,

AND

pulmonary surfactant secretion; phatases; okadaic acid

protein

kinase C; protein

phos-

SURFACTANT enters the alveoli by secretion from type II pneumocytes, facilitates alveolar expansion at birth, and prevents postnatal atelectasis. There is neither a large intracellular nor extracellular reserve of pulmonary surfactant; surfactant secretion is therefore tightly regulated in vivo (31). The phosphorylation and dephosphorylation of proteins are a major mechanism for regulating cellular function that involves two distinct classes of enzymes, termed protein kinases and protein phosphatases (3). Strong evidence exists for a key role of protein phosphorylation PULMONARY

L548

1040-0605/91 $1.50 Copyright

0

and dephosphorylation in the regulation of type II pneumocyte functions such as surfactant secretion. P-Agonists and A2 purinergic agonists stimulate surfactant secretion together with the formation of adenosine 3’,5’-cyclic monophosphate (CAMP) (7, 10, 27). Direct stimulation of adenylate cyclase with forskolin also enhances surfactant secretion with concomitant formation of CAMP and activation of CAMP-dependent protein kinase (17). In vitro phosphorylation of several proteins by CAMP-dependent protein kinase has been examined in rat lung cytosol (30). Phosphorylation of actin by CAMP-dependent protein kinase in rat lung also appears to be developmentally regulated in vivo (29). Surfactant secretion can also be stimulated by calcium ionophores such as A23187 and ionomycin (5, 20), suggesting that surfactant secretion may be regulated by calcium-dependent protein kinases. P, purinergic agonists stimulate surfactant secretion in association with rapid inositol trisphosphate formation and release of intracellular calcium (18,27). Phorbol esters, which powerfully activate the calcium- and phospholipid-dependent protein kinase C, are also potent agonists of surfactant secretion by type II pneumocytes in culture (19). However, the specific molecular mechanisms by which CAMP and/or calcium-dependent protein phosphorylation regulates type II pneumocyte functions such as surfactant secretion are as yet unknown. Involvement of protein phosphorylation by protein kinases in the regulation of type II pneumocyte function also implies a regulatory role for protein dephosphorylation by protein phosphatases. Molecular forms of protein phosphatase (PP-1, PP-2A, and PP-2C) account for almost all the phosphatase activity toward enzymes regulated by phosphorylation on seryl and threonyl residues in the major biosynthetic and biodegradative pathways in liver and muscle, although, in most cases, the contribution of PP-2C is minor (3). Warburton and Cohen (28) have recently described the ontogeny of PP-1 and PP2A activities in developing rat lung. The time course of increasing expression of PP-1 and PP-2A activity coincides with the critical perinatal period of fetal lung ontogeny. Okadaic acid, a polyether derivative of a 38carbon fatty acid marine sponge toxin (24) that is a powerful tumor promoter on mouse skin, neither binds to the phorbol ester receptor nor activates protein kinase C

1991 the American

Physiological

Society

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PROTEIN

PHOSPHORYLATION

IN

TYPE

II PNEUMOCYTES

L549

order to investigate the putative phosphorylation substrates for these PP. Surfactant phosphutidylcholine secretion. Type II pneumocytes were plated at a final density of 2 x lo6 cells/ well in six-well tissue culture plates and incubated overnight in Dulbecco’s modified Eagle’s medium (DMEM; pH 7.4), containing 10% serum and antibiotics with 1.5 &i/well of [3H]choline at 37°C in 95% air-5% CO2 and high humidity. After 20 h of incubation, the cells were washed six times with DMEM, agonists of interest were added, and the cells were returned to the incubator for up to 2 h. After this incubation period, medium was gently collected from the wells, centrifuged at 4,000 g for 3 min to pellet loose cells, and frozen at -70°C after aliquots were removed for LDH assay. Cells were extracted with 0.1% Triton X-100, and extracts were frozen at -70°C after aliquots were removed for LDH assay. Phospholipids were extracted from cells and media (2). “H counts in phospholipid were >98% phosphatidylcholine (PC). Surfactant PC release was expressed as a percentage of [3H]PC in the medium relative to the total amount in cells and medium. In situ labeling of type II pneumocytes in medium containing 32P-labeled inorganic phosphate. Type II pneumocytes were incubated in DMEM containing 1.25 mM CaC12and 0.24 mM Pi (carrier-free inorganic phosphate). The medium also contained fatty acid free bovine serum MATERIALS AND METHODS albumin (40 mg/ml) and 32Pi (250 &i/ml). Incubations Materials were obtained from the following sources: were carried out with 5 x lo6 cells ml-‘. well-’ in sixpathogen-free rats from Charles River Laboratories, Boswell plastic tissue culture plates in 95% air-5% C02, ton, MA; elastase from Worthington Laboratories, Free37OC, and high humidity. Incubation was allowed to hold, NJ; serum from.HyClone Laboratories, Ogden, UT; proceed for 4 h. Steady-state labeling of cellular phostissue culture plasticware from Falcon, Oxnard, CA; tisphoproteins was evaluated by measuring the 32Pspecific sue culture media and other biochemicals of the highest activity of total cellular proteins, which were extracted grade from Sigma, St. Louis, MO. Okadaic acid was a with “stop buffer” [0.2% Triton X-100, 100 mM sodium generous gift from Dr. Y. Tsukitani, Fujisawa Pharmafluoride, 4 mM EDTA, 250 mM sucrose, 0.1% 2-mercapceutical, Tokyo, Japan. toethanol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM Primary culture of type II pneumocytes. Type II pneu- benzamidine, in 58 mM tris(hydroxymethyl)aminomocytes were isolated from male pathogen-free 250- to methane (Tris) . HCl buffer, pH 8.01, at 4OC, precipitated 300-g rats immediately after the lung lavage, elastase with 10% trichloroacetic acid, and the radioactivity was digestion, mechanical dissociation, and immunoglobulin determined by Cerenkov counting. (Ig) differential adherence protocol of Dobbs et al. (6). Two-dimensional electrophoresis of cellular phosphoThis procedure yielded type II pneumocytes with 90% proteins. Mapping of phosphoproteins was carried out viability by trypan blue staining and 95% purity by immediately after the two-dimensional protein electrophosphene 3R fluorescent staining. Cytotoxicity of var- phoresis protocols described by Sinclair and Rickwood ious treatments was assessedby lactate dehydrogenase (2l), as modified from the original paper of O’Farrell (LDH) release into the medium (LDL-20 kit, Sigma), (24). Briefly, isoelectric focusing was carried out in the which was used as a measure of cell integrity. LDH first dimension in tube gels using Biolyte (pH 5-7, Biorelease was expressed as a percent of total cellular LDH Rad, Richmond, CA) as the ampholyte, followed by 5activity. 15% gradient sodium dodecyl sulfate-polyacrylamide gel Pharmacological protocols. Exposure of cultured type electrophoresis in the second dimension. The pH graII pneumocytes to TPA was used to optimize the timedient in the electrofocusing gels was determined by land concentration-dependent activation of protein ki- mm slicing of a blank gel from the same run and measnase C. TPA (10s4 M) for 15 min was then used to urement of the pH of each slice in 0.5 ml deionized water. characterize the protein phosphorylation substrates of Molecular-mass standards were run on the second-diprotein kinase C-dependent phosphorylation in cultured mension gel. The electrophoretograms were standardized by adding identical amounts of protein (25 pg) to each type II pneumocytes. The PP inhibitor okadaic acid was used to determine run. Proteins were identified by silver staining of the gel (Bio-Rad) followed by drying of the gel and autoradiogthe dose- and time-dependent inhibition of PP in cultured type II pneumocytes. Preincubation with 1 PM raphy on Kodal X-OMAT film with image-intensifying okadaic acid was then used to selectively inhibit cellular screens. The films were developed in an X-OMAT auPP-1 and PP-2A in cultured type II pneumocytes in tomatic film processor (Eastman Kodak, Rochester, (22). The purified toxin has been employed to investigate the roles of PP-1 and PP-2A in several physiological systems that are regulated by protein phosphorylation mechanisms (8, 11, 12). The two-dimensional protein electrophoresis techniques of O’Farrell (15) have been used successfully as a method for screening likely targets for regulation by protein phosphorylation and dephosphorylation in cells such as the hepatocyte and neutrophil (9, 16). However, this technology has not yet been applied to the type II pneumocyte, where little is known of the identity of putative regulatory phosphoproteins. In the present study, two-dimensional protein electrophoresis and pharmacological manipulations of protein kinase C and PP activity were used to identify protein substrates of protein kinase C-dependent phosphorylation and PP-dependent dephosphorylation in cultured type II pneumocytes. The phorbol ester 12O-tetradecanoyl phorbol 13-acetate (TPA) was used as a protein kinase C agonist. Okadaic acid, a potent and specific inhibitor of PP-1 and PP-2A, was used as a PP antagonist. Herein, we report several specific phosphoproteins that are substrates for protein kinase C-dependent phosphorylation and PP-dependent dephosphorylation in cultured type II pneumocytes.

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PROTEIN

PHOSPHORYLATION

NY). Computer-assisted two-dimensional densitometric analysis of the phosphoproteins found on autoradiograms was carried out using a Technology Resources (Nashville, TN) system. Measurement of protein kinase C activity. Protein kinase C activity was measured using the methods of Sano et al. (19) for cultured type II pneumocytes. Briefly, protein kinase C activity was measured as incorporation of [y-““P]ATP into histone III-S (Sigma) in cytosol (100,000 g for 60-min supernatant) and in particulates (100,000 g for 60-min pellet) extracted from type II pneumocytes with a solution containing 20 mM Tris HCl, pH 7.5, 10 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2 mM EDTA, and 0.1% Triton X-100. The final reaction mixture (50 ~1) contained 20 mM Tris. HCl (pH 7.5), 5 mM magnesium acetate, 10 PM ATP (X00 cpm/pmol), 10 mM sodium fluoride, and 1 mg/ml histone III-S with and without calcium and phospholipids (2.5 mM CaC12, 40 pg/ml phosphatidylserine, and 0.8 pg/ml diolein). The reaction was linear for at least 5 min and with the addition of up to 5 pg protein. Measurement of PP activity. Phosphorylase a (glycogen phosphorylase EC 2.4.1.1; 1 mol phosphate/m01 97Da subunit) was phosphorylated using phosphorylase kinase (EC 2.7.1.38) and [T-~‘P]ATP was removed as described previously (23). PP-1 and PP-2A are reported to be the only enzymes that exhibit glycogen phosphorylase phosphatase activity in mammalian tissues (13, 14). Therefore, specific PP activity was measured as the dephosphorylation of glycogen phosphorylase a. One unit of PP enzyme activity was defined as the amount of enzyme which catalyzes the dephosphorylation of 1.0 pmol glycogen phosphorylase a/min. PP activity was measured at 30°C in an incubation mixture (30 ~1) containing 50 mM Tris Cl, 0.1 mM EGTA, 0.1% (vol/vol) 2-mercaptoethanol, 5 mM caffeine, bovine serum albumin (1.0 mg/ml), and phosphorylase a (1.0 mg/ml, 10.3 PM). Reactions were initiated by the addition of phosphorylase a after preincubation of the other components for 5 min at 30°C. Assays were carried out for 10 min and terminated and analyzed as previously described (14). Release of radioactivity was limited to ~30% to ensure linear rate of dephosphorylation with respect to time. Assays were performed in duplicate, and control incubations were included in which PP was replaced by buffer. These control values were ~5% of the total radioactivity and were subtracted from the values obtained in the presence of phosphatase. Protamine, which stimulates PP-2A at low concentrations and inhibits at high concentrations and inhibits PP-1 at both low and high concentrations (13, 14), and okadaic acid, which inhibits PP-2A activity with a twolog lower 50% inhibitory concentration (I&) value than PP-1 (28), were used to distinguish between PP-1 and PP-2A activity. When protamine or okadaic acid was present in the assays, they were preincubated with the phosphatase for 10 min at 30°C before the addition of the substrate. The coefficient of variation for the PP results was 85% by 10D6 M okadaic acid. However, the I& concentration for the soluble fraction (lo-’ M) was a log lower than that for the particulate fraction (10D8 M). These findings were also consistent with a predominance of PP-1 in the particulate fraction and PP-2A in the soluble fraction. Okadaic acid was used to inhibit PP activity in cultured type II pneumocytes. The time-dependent effects of okadaic acid on type II pneumocyte PP activity in cultured cells are shown in Fig. 3. Both particulate and soluble fraction PP activity were inhibited in a timedependent manner by 1 PM okadaic acid. By 3 h of incubation, >90% of both soluble and particulate fraction PP activities were effectively inhibited. However, the time to 50% inhibition of soluble fraction PP activity was 1 h, whereas the time to 50% inhibition of particulate fraction activity was 2 h. Significant (>50%) cell detachment from the culture plates was noted after 3 h of exposure to 1 PM okadaic acid. However, no significant

8

0-c

,

-l-4--I 3

I

; 4

in hrs

FIG. 3. Time-dependent effects of okadaic acid on protein phosphatase (PP) activity in cultured type II pneumocytes. Cultured type II pneumocytes were exposed to 1 PM okadaic acid for times up to 4 h. Cells were then extracted, and soluble and particulate fractions were prepared by centrifugation at 100,000 g for 40 min. PP activity in the soluble and particulate fractions were then assayed as described in MATERIALS AND METHODS. A representative experiment from 5 similar replications is shown.

increase in cytotoxicity was observed, as indicated by LDH release. Because of the cell detachment that occurred with exposure of type II pneumocytes to 1 PM okadaic acid for 3 h, it was difficult to assess the effects of PP inhibition with okadaic acid on surfactant secretion using the customary 3-h protocol. However, exposure to 1 PM okadaic acid for 2 h, which did not cause significant cell detachment but caused significant (>75%) inhibition of both particulate and cytosolic PP activity, increased surfactant PC release from type II pneumocytes 55% from 1.2 t 0.2 to 2.2 t 0.5%/h (P < 0.05), whereas coincubation of 1 pM okadaic acid with TPA (10V4 M) for 2 h resulted in an approximately additive increased surfactant PC release to 6.8%/h, 6.5-fold above the resting secretion rate. Two-dimensional protein electrophoresis was carried out to map phosphoprotein substrates of protein kinase C-dependent phosphorylation and PP in type II pneumocytes labeled in culture with “Pi. Several distinct phosphoproteins were identified as putative substrates for protein kinase C-dependent phosphorylation. Autoradiographs of two-dimensional gels showing the location of phosphoproteins separated by isoelectric point in the horizontal dimension and then by molecular mass in the vertical dimension are depicted in Fig. 4. The results of computerized densitometric analysis of these autoradiographs are also displayed in Table 1. Activation of protein kinase C-dependent protein phosphorylation with TPA (10Y4 M) resulted in significantly increased phosphorylation of three specific proteins (50 kDa, p1 5.8 and 5.7; 25 kDa, p1 5.7) within 5 min of exposure to TPA. Phosphorylation of the three proteins was maximal at 15 min and did not diminish significantly in the presence of TPA for up to 3 h (not shown). Phosphorylation of the three proteins was detectable with TPA ( 10V8 M) and increased in a dose-dependent manner to a maximum at (10v4 M), with an EC50 of 10D6 M. Inhibition of >85% total cellular PP activity in the presence of 1 PM okadaic acid for 2.5 h resulted in significantly increased phosphorylation of five specific

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L552

PROTEIN

PHOSPHORYLATION

IN

TYPE

II PNEUMOCYTES

DISCUSSION

25

i0 15 LO

*

15

C

ID

FIG. 4. Autoradiographs of type II pneumocyte phosphoproteins separated by 2-dimensional electrophoresis. Cultured type II pneumocytes were labeled with 32Pi for 4 h. Cells were then treated with agonists of interest: A, control; B, 12-O-tetradecanoyl phorbol 13acetate (TPA, 10m4 M) for 15 min; C, 1 PM okadaic acid (OA) for 2.0 h; n, 1 FM OA for 2.5 h followed by TPA (10e4 M) for 15 min. Protein extracts were first separated by isoelectric point in an electrofocusing tube gel (pH gradient 5-7.7) and then 515% SDS-polyacrylamide gel electrophoresis. Gels were subsequently dried and autoradiographed as described in MATERIALS AND METHODS. Quantitative results obtained by computerized scanning densitometry of gels are shown in Table 1.

1. Results of computerized densitometric analysis of two-dimensional protein electrophoresis patterns of type IIpneumocyte illustrated in Fig. 4 TABLE

Proteins, kDa: p1: TPA (1O-4 M) OA (IO-” M) OA + PMA

50 5.8 50

5.1

-

45

-

40

5.6

5.5

5.5

1,500 3,080

805

708

180

5.7

5.5

55

80

200

25

1,010

838

100 145

Results are expressed as percentage increases in area density units. Respective effects of phorbol ester 12-0-tetradecanoyl phorbol 13acetate (TPA) (Fig. 4, B), okadaic acid (OA) treatment (Fig. C), and combined OA and TPA treatment (Fig. 40) are compared with densitometry of control unstimulated cells (Fig. 4A). The experiment was repeated 6 times with similar results. Only statistically significant (ANOVA) mean differences in area density have been tabulated (P < 0.01, n = 6).

proteins (50 kDa, p1 5.7 and 5.6; 45 kDa, p1 5.5; 40 kDa, ~15.5; 25 kDa, ~15.5). Preincubation with 1 PM okadaic acid for 2.5 h followed by exposure to TPA (10e4 M) resulted in additional significant phosphorylation of four specific proteins (50 kDa, p1 5.6; 4.5 kDa, p1 5.5; 40 kDa, ~15.5; 25 kDa, ~15.5). Similar results were obtained with 2-h preincubation with 1 PM okadaic acid (not shown). We conclude that protein kinase C-dependent phosphorylation and PP-dependent dephosphorylation of specific phosphoproteins occurs in type II pneumocytes in culture in response to pharmacological manipulation of the respective regulatory systems. We further speculate that phosphorylation and dephosphorylation of the type II pneumocyte proteins mapped herein may play a role in regulating type II pneumocyte function.

Basal surfactant secretion from unstimulated type II pneumocytes in culture was similar to that reported previously (5, 7, 10, 17-20, 27, 29, 30). Translocation of protein kinase C with increased activity in the particulate fraction agreed with the results of Sano et al. (19). Although the stability of the type II pneumocyte phenotype in culture is controversial, the present study presents new direct molecular evidence supporting the concept that protein kinase C-dependent phosphorylation and PP-dependent dephosphorylation of specific phosphoprotein substrates play a role in the functional regulation of type II pneumocytes in culture. PP-1 is inhibited, but PP-2A is stimulated, by low concentrations of protamine (13, 14). In addition, okadaic acid inhibits PP-2A activity at a two orders of magnitude lower concentration than PP-1 (4). On the basis of these criteria, the results of the current study indicate a predominance of PP-1 activity in the particulate fraction of type II pneumocytes and a predominance of PP-2A activity in the cytosolic fraction. These results are in agreement with the reported distribution of PP-1 and PP-2A activity in whole adult, neonatal, and fetal rat lung (28). Inhibition of PP-1 and PP-2A with okadaic acid results in increased phosphorylation levels of numerous phosphoproteins in hepatocytes and adipocytes, together with functional effects such as stimulation of glycogenolysis and gluconeogenesis in hepatocytes and the conversion of glucose to glyceride-glycerol in adipocytes (16). In the current study, inhibition of PP activity with okadaic acid resulted in highly significant increases in phosphorylation of five proteins, with the formation of clearly visible acidic charge trains. Although the identity of these proteins is presently unknown, it is tempting to speculate that they may be involved in the regulation of type II pneumocyte function. The cell detachment observed in the presence of PP inhibition with okadaic acid points to the involvement of cytoskeletal regulatory elements. Further studies will be necessary to elucidate the effects of PP blockade with okadaic acid on the phosphorylation of cytoskeletal elements and cell adhesion in general. Stimulation of protein kinase C translocation from the cytosol to particulate fraction with TPA also resulted in significantly increased phosphorylation of three proteins in type II pneumocytes. Because this effect was associated with the well-recognized agonist effect of TPA on surfactant PC secretion, it is tempting to speculate that these phosphoproteins are involved in the regulation of surfactant secretion. One of the proteins that was phosphorylated in response to PMA (50 kDa, ~15.7) was also phosphorylated, but to a greater specific activity, in response to PP inhibition with okadaic acid. This protein may present fruitful opportunities for study of the functional overlap between protein phosphorylation and dephosphorylation in the regulation of type II pneumocyte function. CAMP-dependent in vitro phosphorylation of type II pneumocyte cytosolic proteins with molecular masses of 260, 240, 44, and 22 kDa has been reported by Whitsett and co-workers (30). Actin (43 kDa) was reported to be

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PROTEIN

PHOSPHORYLATION

the most abundant substrate for in vitro CAMP-dependent phosphorylation both in rat lung cytosol as well as in type II pneumocyte cytosol, and its in vitro phosphorylation in rat lung cytosol was reported to be developmentally regulated during the perinatal period of fetal lung ontogeny (29). In the current study, type II pneumocytes were labeled with 32Pi in culture. Proteins with molecular masses of 40 and 45 kDa were not significantly phosphorylated in resting cells in culture but were highly phosphorylated in response to PP inhibition with okadaic acid. Additional phosphorylation of these proteins was induced by TPA following okadaic acid. Thus patterns of protein phosphorylation in type II pneumocytes in culture differs from in vitro protein phosphorylation patterns. In addition, the phosphoprotein substrates of protein kinase Cdependent phosphorylation and PP-dependent dephosphorylation reported herein appear to differ from the reported in vitro substrate proteins of CAMP-dependent phosphorylation in the type II pneumocyte (29, 30). The putative phosphoprotein substrates of protein kinase C and PP reported herein are of low relative abundance among type II pneumocyte proteins. Substrates of protein kinase C-dependent phosphorylation with respective molecular masses of 63, 23, and 21 kDa have been reported in secretory granules of adenohypophysial cells (25). A protein kinase C substrate with a molecular mass of 47 kDa has also been reported in platelets (26). An 87-kDa substrate of protein kinase C has been purified from brain and is present in synaptosomes (1). It has been speculated that these protein kinase C substrates may play roles in the regulation of secretion in the respective systems, but their function is incompletely characterized. Ongoing research is designed to provide positive identification of the putative phosphoprotein substrates described herein, based on micro-amino acid sequencing. Amino acid sequence site-specific phosphorylation analysis will also be necessary to further elucidate the molecular role of these phosphorylation and dephosphorylation reactions in functional regulation of the type II pneumocyte. We conclude that the specific phosphoproteins described herein comprise major substrates for protein kinase C-dependent phosphorylation and protein phosphatases in type II pneumocytes from rat lung studied in culture. We speculate that these phosphorylation and dephosphorylation reactions will prove to play key roles in the regulation of type II pneumocyte function. D. Warburton thanks Philip Cohen, Grahame Hardie, and Elizabeth Carrie of the Medical Research Council Protein Phosphorylation Group at the University of Dundee, Scotland, for helpful advice and encouragement at the inception of this project and Frederick L. Hall for reading the manuscript. This work was supported in part by a grant from the American LungAssociation of Los Angeles County and by National Heart, Lung, and Blood Grant ROl-HL-44060 to D. Warburton. Address for reprint requests: D. Warburton, Div. of Neonatology and Pediatric Pulmonology, Children’s Hospital of Los Angeles, Bin 83, 4650 Sunset Blvd., Los Angeles, CA 90027. Received

21 June

1990; accepted

in final

form

27 November

1990.

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L554

PROTEIN

PHOSPHORYLATION

23. STEWART, A. A., B. A. HEMMINGS, P. COHEN, J. GORIS, AND W. MERLVED. The Mg-ATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities. Eur. J. Biochem. 115: 197-205,198l. 24. TACHIBANA, K., P. J. SCHEUER, Y. TSUKITANI, H. KIKUCHI, D. VAN ENGEN, J. CLARDY, Y. GOPICHAND, AND F. J. SCMITZ. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Halichondra. J. Am. Chem. Sot. 103: 2469-2471,198l. 25. TURGEON, J. L., AND R. H. COOPER. Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules. Biochem. J. 237: 53-61, 1986. 26. TYERS, M., R. A. RACHUBINSKI, C. S. SARTORI, C. B. HARLEY, AND R. J. HASLAM. Induction of the 47 kDa platelet substrate of protein kinase C during differentiation of HL-60 cells. Biochem. J.

IN

TYPE

II PNEUMOCYTES

243: 249-253,1987. 27. WARBURTON, D., S. BUCKLEY, AND L. COSICO. P, and Pp purinergic receptor signal transduction in rat type II pneumocytes. J. Appl. Physiol. 66: 901-905, 1989. 28. WARBURTON, D., AND P. COHEN. Ontogeny of protein phosphatases 1 and 2A in developing lung. Pediatr. Res. 24: 25-27, 1988. 29. WHITSETT, J. A., D. W. HULL, C. DION, AND J. LESSARD. CAMP dependent actin phosphorylation in developing rat lung and type II epithelial cells. Ex~. Lung Res. 9: 191-209, 1985. 30. WHITSETT, J. A., S. MATZ, AND C. DAROVEC-BECKERMAN. CAMPdependent protein kinase and protein phosphorylation in developing rat lung. Pediatr. Res. 17: 959-966, 1983. 31. WRIGHT, J. R., AND J. A. CLEMENTS. Metabolism and turnover of pulmonary surfactant. Am. Rev. Respir. Dis. 135: 426-444, 1987.

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Protein phosphorylation and dephosphorylation in type II pneumocytes.

Protein phosphorylation and dephosphorylation are a major mechanism for regulating cellular activity. Substantial evidence exists for ascribing a key ...
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