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Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells
ELSEVIER
Biochimicaet BiophysicaActa 131 I ( 1996~ I 17-123
Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells Lln-Jin P. Zimmerman " Mengshu Wang, June B. Nelson, Felix S. Ekwunife, Lin Liu blstitute fiJr Em'ironmental Medicine. Unicersitv t~ Pennsyh ania Medical Ce,lter. Philadelphia. PA 19104. USA
Revi~ed 12 October 1995:accepted6 No,ember 1995
Abstract Stimulation of secretion from rat alveolar epithelial type il cells by the fl-adrenergic agonist terbutaline activates cAMP-dependem protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type 11 cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within I-3 rain of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppres~ble by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two er,dogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for ~cretion. Keywords: Pneumocytetype 11:Terbutaline: Protein kinase A: Calpain
1. I n t r o d u c t i o n Two phenotypically different epithelial cells, type ! and II, line the pulmonary alveolus [1]. Type n cells synthesize lung surfactant which is stored in lamellar bodiex and secreted by exocytosis [2-4]. Previous studies have shown that the secretory activity of type II cells can be enhanced by various secretagogues such as calcium ionophore, parinergic and ~-adrenergic agonists [5,6]. Stimulation of type II cells, for example, with the ~-adrenergic receptor agonists terbutaline or isoproterenol activates adenylate cyclase via G-protein and results in increased cAMP levels that activate PKA [7,8]. Activated PKA then phosphorylates specific protein(s) presumably involved in the secretory processes [9]. Recently we observed that the same set of secretagogues also activates endogenous calpain in isolated alveolar type It cells [10]. Furthermore, type iI cells pretreated with a cell-permeable calpain inhibitor exhibited substantially depressed ( 4 0 - 5 0 % ) secretory ac• Correspondingauthor. Fax: + I (215) 8980868. 01674889/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0167-4889(95)00181-6
tivities [11]. In this study, therefore, we investigated the effect of calpain activation on protein kinase A and its phosphorylation activity in alveolar type I1 cells stimulated with terbutaline. Calpains (EC 3.4.22.17, calcium-activated neutral proteases) are characterized by their ability, :e perform limited proteolysis of specific proteins including protein kinases [12]. cAMP-dependent protein kinase (PKA) [13.14], calcium-phospholipid-dependent protein kinase (PKC) [ 15,16], and pp60 ~r~ tyrosine protein kinase (TPK) [17] are cleaved by calpain. Proteolytically modified protein kinases are functionally altered and can phosphorylate without cofactors. For example, upon binding cAMP, PKA is activated when tts regulatory subunits dissociate from the enzyme. The regulatory subunits of PKA are, however, susceptible to proteolysis by calpain and can be cleaved into 39 and 17 kDa fragments [13]. The proteolyzed regulatory subunit is then no longer able to reassociate with the catalytic subunits to inhibit them and the enzyme remains activated without cAMP [14]. The regulatory fragments, however, retain their ability to bind cAMP and can be photolabeled
U..J.P. Zimmerm,net ul. / Biochhnicaet Biophysh'aActa 1311 t lqV6) 117-123 with 8-N~-[ ~'P]cAMP. a photoaffinity analog of" cAMP [18,19]. cAMP-binding (35-39 kDa) proteins are produced following the endogenous [I 3] or exogenous [20.21] proteolysis of regulatory subunits from both PKA isozymes. These fragments are apparently formed in vivo since their levels are not reduced following homogenization of tissues in the presence of EDTA or other protease inhibitors [14.22.23]. Age-dependent changes in the amount of endogenously proteolyzed PKA regulatory subunits in mouse lung are also reported [24], In the present study, we investigated whether proteolysis of PKA by calpain occurs and engenders sustained phosphorylation activity in stimulated type II cells. The effect of proteolysis on the stimulus-dependent phosphorylation activity of PKA was examined utilizing the colpain inhibitor 11 (N-acetyl-leucyl-leucyl-methioninal). This inhibitor is known to possess good membrane penetratability and is a potent inhibitor of calpain [11,25,26]. The stimulus-induced proteolysis of PKA was monitored by photoaffinity labeling of the proteolyzed fragment. Endogenous proteins specifically phosphorylated by activated PKA were identified by their responses to calpain or PKA inhibitor.
2. Materials and methods
All culture material was purchased from GIBCO Laboratories (Grand Island. NY), [y-3-~P]ATP (2 mCi/ml: 3 Ci/mmol) and [~z P]orthophosphate (1 mCi/ml) were purchased from Amersham (Arlington Heights, IL). 8-N 3[3_,P]cAMP (8-azidoadenosine-3'.5'-cyclic monophosphate. I mCi/ml; 50 Ci/mmol) and monoclonal anti-actin (C4MAb) were purchased from ICN (lrvine, CA). Calpain inhibitor II (N-acetyl-leucyl-leucyl-methioninal) was purchased from Boehringer Mannheim (Indianapolis, IN). Kemptide (cAMP-dependent protein kinase substrate. LRRASLG) and phosphocellulose units for PKA assay were purchased from Pierce (Rockford, IL). Synthetic cAMPdependent protein kinase inhibitor (rabbit sequence TTWADFILASGRTGRRDAILHD), terbutaline, charcoal (at,,ivated) and other chemicals were from Sigma (St. Louis. MO). 2.1. Isolation of ah'eolar epithelial O'pe H cells and preparation ~f c:vtosol fraction Alveolar type 11 cells were isolated from rat lung, as previously described by Dobbs at al. [27]. Isolated type ii cells were plated at 10 million density in 100 mm cell culture dishes (Costar). After 18-20 h of culture in minimal essential medium (MEM) with 10% fetal calf serum (FCS) and in air/CO, (19:1), the medium containing unattached cells was discarded. Adhering type II cells (3-4 million. 30% plating efficiency) were equilibrated further with fresh MEM without FCS or prcincubated wiih MEM
containing 10 /zM of calpain inhibitor II for 45-60 min before initiation of experiments. Inhibitor treated or untreated cells (3-4 million) were subsequently stimulated with terbutaline (10 ,aM) for 0, 1, 2, 3, and 5 min. Control cells (0 rain) had no additions. At the end of each incubation period, activated charcoal (0.3 m g / m l ) was added and the medium was quickly removed. The cells were wa:,hed twice with ice-cold PBS containing I mM EGTA and then scraped gently. Harvested cells were pelleled by cemrifugation at 1000 rpm for 10 rain. The cell pellet was lysed in 100-150 ,al of ice-cold lysing buffer (40 mM Tris, 150 mM NaCI. 2 mM EGTA, 10 mM Chaps. I mM PMSF, I0 mM benzamidine, 0.1 mM leupeptin. 0.1% aprotinin. 0.1 ,aM pepstatin, 0.Irk NAN,, pH 7.0) for 60 rain and further distrupted by sonication (Ultrosonicator. Inc., setting 7) for 5 s twice. Cytosolic supernate was used for PKA phosphorylation activity determination and photoaffinity labeling. 2.2. Assay for phosphorylation actirity of PKA A reaction mixture (50/J,I) containing cytosolic fraction (30-40 ,ag), MgCI 2 (5 mM), ATP (5 ,aM) and Kemptide (40 ,ag) was used for assay of PKA phosphorylation activity. For the PKA inhibited samples, synthetic PKA inhibitor (10 ,aM) was also included. Alter a 3-min preincubation, the reaction was started by adding [3"~P]ATP (2.5 x 106 cpm). Phosphorylation was terminated after 10 min by the addition of an equal volume of stopping buffer (50 mM Tris, 10 mM NaF, I mM DTr, pH 7.6). The reaction mixture was spotted on a phosphocellulose microcentrifuge unit and centrifuged for 30 s. The adhering sample was washed with 250 /1,1 of phosphoric acid (75 mM) and centrifuged. The process was repeated. The phosphocelluse unit containing the processed sample was transferred into a scintillation vial and counted. 2.3. Assay for endogemms proteins phosphorylated by PKA The reaction mixture contained the cytosolic fraction and MgCI,. with or without synthetic PKA inhibitor (10 p,M). The reaction was initiated by adding [~-'P]ATP. Following a 10-min incubation, phosphorylation was terminated by adding solubilizing buffer. The mixture was then heated at 100°C for 5 min and subjected to SDS-PAGE analysis lbllowed by autoradiography. The radioactivity of selected protein bands stained with Coommassie blue was scanned by AMBIS. 2.4. Metabolic htbeling ~f alceolar type H ceils with H: P]orthophosphate After 1~-20 h of culture in MEM with 10% FCS. type II cells plated at I0 million density in 100 mm cell cuture dishes were washed with phosphate-deficient MEM l\,r 6
U.-,I.P. Zmmu', mm et ,I. / Ri,chimica el Bioph~~i~a.,h ta 1311 ¢ItJt)fil I 17-123 times and then incubated with [~ZP]orthophosphate (10 / x C i / m l ) for 3 h. Labeled cells were either pre-treatcd with 10 /aM calpain inhibitor 11 for 60 min or left untreated. Both types of labeled cells were subsequently stimulated with terbutaline (/.aM) lbr 5 rain. The cells were harvested and solubilized in sample buffer and analyzed by S D S - P A G E and autoradiography for endogenously phosphorylated proteins. 2.5. Photoaffbti~." labeling PKA
~ .,:,: _ ~ o ~ 2::-
~,
~5. :'_ 2co
of the regtdatory sttlmnit o f
aliquot (100 p.l, I m g / m l ) of cytosolic fraction was placed ip a microtiter plate well containing a small bar magnet. Photolabile radioactive c A M P (8-N~-[ ~-~P]cAMP. 1 /aM) was added and incubated for 30 min in the dark at r o o m temperature. Photolysis was carried out using a U V S - 5 4 mineralight l a m p 10 c m from the plate for 15 min at r o o m temperature. An aliquot (50 # l ) o f the photolyzed sample was injected into an equal v o l u m e of 5 ~ T C A for determinat;on o f T C A precipitable radioactivity. Another aliquot (50 /zl) was injected into the stopping buffer (50 mlVl Tris. pH 7.6, 10 m M NaF. 1 m M D T ' I ) for analysis of photolabeled proteins by S D S - P A G E and autoradiography.
~ ~:-
An
2.6. Other procedures S D S - P A G E was carried out according to L a e m m l i [28] using 7.5 or 10,% gel. Protein concentrations were determ i n e d by the m e t h o d o f Bradford [29] using bovine 3'globulin as a standard.
c~a
: 3
7
3
2
a
5
Time (min)
Fig. I. The cllccl ,ff calpain inhibition on the stimulus-induced PKA pho~,phor~,l,,li,m ,cli,.it) in alveolar epithelial type n cells. Cells ',,.ere either pretreated :'.ith calpain inhibitor I1 (N-acetyl-leucyl-leucyi-methioninal. II) #M) lor 45-60 min or left untreated. Both types of cells were then ,timulatcd '.,.ith tcrbutaline ( I0/aM) for 0. I. 2.3.5 rain. Cell lysate (30-40/zg) '.'.a~a,,,a'.ed for PKA acti'.ily ,asing Kemptide (40 #g) as the ,ub-trate v.ith or v.ithout s.'.nthetic PKA inhibitor ( I0 #M) as, de:~,cibedin Sccdon 2. [ '-"P] incorporation {pmol/mg protein) wa:~,plotted a~. lunction of qimulatioll time. ~. Untreated cells: A. assayed in the presence of PKA inhibitor: O. cells pre-treated with calpain inhibitor; z~. assayed in the pre,encc of PKA inhibitor. Values are means±S.E, of 4-6 experimcnt~
A
10
-
97 -
3. R e s u l t s
3.1. Effect o f calpain rm P K A a c t i r # v in ~.'pe II cells in response to stimulation T o investigate the effect o f stimulus-induced calpain activation on the P K A activity in type 11 cells, P K A activities o f the cells pretreated with calpain inhibitor were c o m p a r e d with their untreated counterparts. Phosphorylation activity in the calpain-inhibited cells should represent native P K A activity without proteolytic modification. Thus, cells w e r e preincubated with cell-permeable calpain inhibitor II (N-acetyl-leucyl-leucyl-methioninal. 10 ,ttM) for 4 5 - 6 0 min prior to stimulation by terbutaline for 0. I. 2, 3. and 5 min. The inhibitor concentration and preincubation time were previously d e t e r m i n e d by the inhibition o f [3H]phosphatidylcholine secretion from type II cells prelabeled with [3H]choline [1 I] and by inhibition of proteolysis o f e n d o g e n o u s spectrin in intact type II cells [ 1 0 ] P K A activity o f the cytosol fraction was assayed using PKA-specific substrate (synthetic peptide, Kemptide). Fig. 1 indicates that P K A activity rises to a m a x i m u m within 1 - 2 min and thereafter declines during 3 - 5 min o f stimu-
64
!'14-
4 k 1
2
3
4 1
2
3
4
Fig. 2 Photoatlinit) labeling of PKA regulatoo ~,uhunits in type n cells. Acti'~atcd charcoal treated cell ly:.,ate fi'om type II cells stimulated with terhutaline t 10 /z~.l~ Ibr 0. 2. 3 min were photoaffinity-labeled v,ith ;-;-azido-cAMP ( I p..M) as described in Section 2. A: TCA precipitable radioacti~il.~. Aliquot of photolysed mixture was precipitated by equal ,.olume 5c~- "[CA and counts of the precipitate ~erc plotted as function of ~timulation time. Values sho'.,,n are averages of tv,o experiments. B: Photolabeled prolcm~. Aliquot of photolabeled mixture was analyzed by SDS-PAGE (7.5'~ gel) t'~llowed by autoradiography. Lane I. unstimulated celh,: lane 2. 2 rain: lane 3. 3 rain r.timulated cells: lane 4. calpain inhibited ,:cll~.
I,'.-J.P. ZintmelTnan et al. / Biochimictt et Biophysica Acta 1311 19961 117-123
112 97 -
:.,
:.
~mb~mD .,,imp, ~ 1
2
3
~4111.,,imB ¢lmm,.ml 4
5
6
7
8
Fig. 3. Endogenous proteins phosphorylated by PKA in type II cells stimulated b) terbutaline. Cells v.ere stimulated with terbutaline for O, 1. 2. 3 min. Cytosolic fractions (30 /zg) in lysing bufl~r (see Section 2) were incubated with MgCI. (5 raM) and ATP (5 /.tM). For the PKA-inhibited samples, synthetic PKA inhibitor ~I0 ~M) was also present. Alter 3 min preincubation, the reactkm was stalled by addition of [~"PIATP (2.5×10 ~ cpml. After 10 min incubation, the reaction mixture was directly solubilized in sample buffer and analyzed by SDS-PAGE (7.5cb gel) followed by autoradi[zraphy.Lanes (I-4): 0. 1.2.3 min stimulatnon. I~mes (5-8k O. I. 2. 3 min stimulation and assayed in the presence of PKA inhibitor.
type II cells in response to stimulation, the appearance of this fragment was monitored by photoaffinlty labeling. The fragment was covalently labeled wdh a photolabile analogue of radioactive cAMP. 8-azido-cyclic-[ ~2P]AMP. The fragment binds photolabel with high affinity ( 1 2 5 - 2 0 0 nM) and exhibits the same binding characteristics as c A M P [30]. Photolysis of 8-N-[ 32P]cAMP by UV (245 nm) generates a nitrene group which ~:an react with any amino acid residues within its binding site. Isolated type II cells ( 3 - 4 million/plate) were stimulated with terbutaline (10 ~ M ) for 0. 2 or 3 rain. The cytosolic fractions were then preincubated with 8-N 3[ 3 : p ] c A M P (I g,M) for 30 rain in the dark and subsequently photolyzed using a UVS-54 mineralight lamp for IO rain (see Section 2). Aliquots were withdrawn for the determination of TCA precipitable incorporation of radioactive photolable and for analysis of the photolabeled proteins by S D S - P A G E and autoradiography. Fig. 2A indicates that T C A precipitable radioactivity increased during the first 2 - 3 min of stimulation. Analysis of the photolabeled proteins by S D S - P A G E and autoradiography revealed that in the unstimulated cell lysate, the P K A regulatory subunit type 1 (RI, 49 kDa) alone was heavily photolabeled, while the regulatory sub-
" q 112
lation (n = 6), Protracted stimulation of 3 0 - 6 0 min yielded no delayed additional activity. The initial PKA activity of
unstimulated cells (200 pmol/mg protein) increased 1.52.0 fold (367 pmol/mg), reaching a maximum at 2 min
declining to half (260 pmol/mg) by 3-5 rain. The rise and fall of PKA activation was also observed in the calpain-inhibited cells except that m a x i m u m activity occurred consistently earlier (I min) and was 2 2 - 2 5 % lower than the untreated cells (n = 6). When synthetic PKA inhibitor was included in the assay mixture, stimulus-dependent modulation of phosphorylation activities was absent and was 7 0 - 7 5 % less than the m a x i m u m activity of either untreated or inhibited cells. The data suggest that stimulation of type II cells with terbutaline activates calpaiu and PKA, and that calpain up-regulates maximal P K A activity for 1 0 - 1 5 % and prolongs it relative to calpain-inhibited cells.
66-
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!i!¸m 21-
....
3.2. T k e s t i m u h t s - i n & . ' e d cells
proteolysis o f P K A in ~'pe H
Previous study indicated that trypsin (or an endogenous calcium-dependent protease during purification) cleaved the regulatory subunit of PKA isozyme II and generated a distinctive 39 kDa fragment which is still able to bind c A M P [13]. To confirm the limited proteolysis of P K A in
1
2
3
4
5
6
7
8
Fig. 4. The effect of calpain inhibition on the phosphorylation of endogenous proteins in type II cells stimulated by terbutaline. Cells were either pre-treated with calpain inhibitor 11(10 #M) far 60 min or left untreated. Both type of cells were then stimulated with lerbutaline (IO /.tM) for 0, I, 2.3 rain. Cytosolic fractions t60/,~g) was incubated with phosphorylation reaction mixture and processed as described in Fig. 3 legend. Lanes ( I-4): 0, I. 2, 3 rain stimulation of untreated ceils. Lanes (5-8): 0,1, 2, 3 rain stimulation of calFain-inhlbitedcells.
U *J.P. Zimmenmm et al. / Bio¢himi¢ ,~ ¢'t Itiol,hv~i~, A,t,': / ¢! / ~/"961 I /7-123
3.3. E n d o e e m m s p r o t e i n s p h o s p h o r y l a t e d
Pl10
]I c e l l s in r e s p o n s e to s t i m u l a t i o n
100 ;
T o identify the e n d o g e n o u s proteins phosphorylated by PKA in response to stimulation, the standard P K A assay was carried out without Kemptide. Assays were also performed ~ith and without synthetic P K A inhibitor. T h e samples were subjected to S D S - P A G E and auloradiography. From visual estimates o f the intensity o f phosphor vlated protein hands, two proteins with electrophoretic mobility corresponding to 112 and and 47 k D a were j u d g e d to be the e n d o g e n o u s substrates o f P K A . T h e stimulus-dependent phosphor3,1ation kinetics o f these proteins was ( I ) similar to the activity profile observed in vitro (Fig. 3. lanes I - 4 and Fig. 4. lanes I - 4 ) . (2) abolished when P K A inhibitor was present (Fig. 3. lanes 5 - 8 ) . and (3) modulated when calpain inhibitor was present (Fig. 4. lanes 5 - 8 ) . Furthermore, when e n d o g e n o u s phospho~,lation of 3:P metabolically labeled cells was examined, proteins (104, 138. I I 0 , 84. 73, 53, 47, 40 and 29 kDa) were phosphorylated in response to a 5-min slimulaTitm xsith terbutaline. A m o n g these proteins, p110
6o~
o
1 40 -;i
d
by P K A in O'pe
120
i n,"
121
i
0
P47 18
14 -; Z
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:
~ 0
a
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6
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,'.
~-i
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i
01
'
66
-
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3
Fig. 5. In vivo phosphoo'lation of endogenou,~ proteins in phosphorous-32 labeled alveolar type 11 cells stimulated with terbutaline, lsohacd type II cells were labeled with [~:Plorthophosphate (IO p-Ci/n'd) for 3 h. Labeled cells were either pre-treated with calpain inhibitor 11( 10 ~M) for 60 min or left untreated. Both types of labeled cells were subsequentl? stimulated with terbutaline (10 p,M) for 5 min. Cell~, v.ere harse~ted. solnbilized in sample buffer and analyzed by ~DS-PAGE and autoradiography. The radioactivities in p112 and 1347protein hands '.','erescanned by AMBIS and plotted. (I) Unstimuluted cells. (2) (erbutuline-stimulated cells. (3) cells pre-treated with calpain inhibitor and then stimulated with terbutaline.
unit type II (RII, 56 kDa) and a proteolytic fragment o f R (39 kDa) w e r e only faintly photolabeled. In the stimulated cells, h o w e v e r , all three were intensely photolabeled. In addition, a 45 and 43 k D a doublet and a 33 k D a protein were also labeled (Fig. 2B). A radioscan indicated that photolabeling o f the 39 k D a f r a g m e n t increased 6- to 8-fold in the stimulated cells ( n = 2). T h e results indicate that P K A is subject to proteolysis in type 11 cells in response to stimulation and that proteolysis is complete within 2 - 3 min.
45-
~O
~
47
:~,~
21-
1
1
i
W
~
2
3
4
Fi~. 6. Endogenott~ proteins phosphorylated in type II cells stimulated by terbulatine and l~.,a',ed in the presence of exogenous cAMP. Cell lysate from cell, stimulated with terbutaline for O, I. 2. 3 min was assayed for PKA activity in the presence of cAMP (10 /zM) and analyzed by SDS-PAGE ( 10% gel) followed by autonldiogruphy. Lanes ( I ) 0 rain. (2) I min. (~) 2 min. (4) 3 min.
U..Z P. Zimmcrman ,'t al. / Biochimica el Biophysica Acta 1311 t 1996) I 17-123
and p47 showed 20-25c/~ reduced phosphorylation in calpain-inhibited cells relative to uninhibited cells (Fig. 5). The specificity of the endogenous proteins phosphorylated by PKA was further demonstrated by a PKA assay in which exogenous cAMP (I /.tM) was added. Under this condition, the phospho"vlation of p47 was highly enhanced relative to the other proteins (Fig. 6). Thus. the p47 protein appears to bt: the major target of activated PKA in type II cells in response to stimulation by terbutaline.
4. Discussion Enhanced secretory activity of alveolar epithelial type I1 cells is associated with an increased intracellular cAMP (or calcium) concentration and activation of protein kinase PKA (or PKC). Intracellulal cAMP (or calcium), however, rises only transiently, whereas observable enhanced secretion occurs much later at 1-2 h [5]. How the transient rise in the second messengers and subsequent activation of protein kinases relate to the augmcoted secretory activity of type II cells over longer time periods remains obscure. Furthermore. the cellular proteins phosphorylated by activated protein kinase have not been well characterized an~t their exact role in the secretory activity of type 11 cells is presently unknown. In contrast to neurotransmitier release or platelet activation. stimulated secretion of type I1 cells is a relatively slow and protracted process that may require the sustained elevation of second messenger or signal-transducer activity. It is not known whether the process is effected by a prolonged protein kinase activity or by multiple repetitive activations in time. We examined therefore whether the limited proteolysis of PKA by activated calpain might provide a plausible mechanism for sustained augumentation of secretion. Proteolytically modified PKA is known to remain activated without initial activating cofactor cAMP [141. The results of our photoaffinity labeling experiment indicate that limited proteolysis of PKA indeed takes place in type II cells in response to stimulation. It has been shown that trypsin treatment of purified PKA regulatory subunits R I (49 kDa) and R n (56 kDa) results in 37 and 39 kDa fragments, respectively [211. To our knowledge, a comparable study using calpain has not been done. In our own cell system, however, other protein bands between 49 and 39 kDa are also photolabeled. In addition, the photolabeling of the full-length regulatory subunits (56 and 49 kDa) increases in spite of increased proteolysis in response to stimulation. The reason for enhanced photolabeling of the full-length regulatory subunits is not clear. It has been shown that regulatory subunits exist in either holoenzyme form or dissociated state, and that the relative proportion of each may change, for example, with age [24]. A stochastic change in proportionality in response to stimulation
may favor the dissociated subunit with higher affinity for cAMP and thereby result in increased photolabeling. In parallel with proteolysis, PKA activity appears to be sustained at elevated levels longer than calpain-inhibited cells. The peak activities of both cell groups are nevertheless transient, leveling off after 5 rain of stimulation with no later peaks. Hence. our results suggest that secretory activity of type II cells is sustained by continuous augmented activation rather than multiple repetitive activation by PKA. The proteins p112 and p47 proteins specifically phosphorylated by PKA in response to stimulation are potential signal transducers for type II cell secretion. In view of the highly enhanced phosphorylation of p47 protein in the presence of exogenous cAMP. it appears to be the preferred substrate of PKA. Our preliminary studies by immunoblot with monoclonal antibody anti-actin followed by autoradiography, however, suggest that the p47 protein is not an actin molecule. Under our experimental conditions. actin itself was not phosphorylated in type It cells in response to terbutaline, in contrast to a previous study reporting cAMP-dependent phosphorylation of actin in type II cells [9]. In conclusion, stimulus-induced PKA activity is up-regulated and sustained by calpain. The target of PKA activity appears to he two proteins, p112 and p47. The identity of these proteins remains to be elucidated.
Acknowledgements The authors are grateful to Dr. George A. Hermann for his editorial help and to Mrs. Elaine Primerano for the preparation of the manuscript. This work was supported by U.,~. Public Health Service Grant ROl HL49982 (UJ.Z.).
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(Mellgren, R.L. and Murachi, T. eds.), pp. 3-24, CRC Press, Boca Raton. FL. [13] Corbin, J.D., Sugden, P.H., West, L.. Flockhart. D.A., Lincoln. T.M. and McCarthy. D. (1978)J. Biol. Chem. 253. 3997-4003. [14] Weber. W. and Hilz, H. (1978) Eur. J. Biochem. 83, 2 1 5 - 2 2 5 [15] Kishimoto, A., Kajikawa, N,, Shiota, A. and Nishizuka. Y. (1983)J. Biol. Chem. 252. 7610-7616. [16] Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda. I.. Tanaka. S.. Tominaga. M., Kuroda, T. and Nishizuka, Y. (1989) J. Biol. Chem. 264, 4088-4092. [17] Oda. A., Drukers. B.J.. Ariyoshi. H.. Smith. M. and Salzman. E.W. (1993) J. Biol. Chem. 268. 12('q3-12608. [18] Farago. A.. LeHang, P., MeszartJ:;, G. and Antoni. F. (1980) Biochim. Biophys. Acta 632.6,1,9-660. [19] Haley, B.E. (1977) In: Methods in Enzymology (Jakoby. W,B.. and Wilchek, M.. eds.). Vol, 4I, pp 339-346. Academic Press, Ne~ York. [20] Potter. R.L. and Taylor. S.S. (1979) J. Biol. Chem. 254. 2413-2-1.18
[2 I] Potter. R.L. and Taylor, S.S. (1979) J. Biol. Chem. 254. 9000-9005. [22] Ima,huku. 1.. Fossett. M.C.. Ill and Green. A.A. ( 19791 Cancer Res. 3LL 3006-3013. {23] Pamer. S.S.. Butley. M.S. and Malklnson. A.M. (19811 J. Neurochem 36. 2080-2085. [24] Beer. D.G.. Butley, M.S. and Malkinson. A.M. (198D Arch. Bkyzhem. Bit,phys. 228. 207-219. [25] Kwak. K.B.. Kambayashi. J.-I., Kang, M.S. Ha. D.B. and Chung. C.H. (19931 FEBS Lett. 323. 152-154. [2t.] N1ehdL S. Angelastro. M.R.. Wiseman. J.S. and Bey. P. (1988l Biochem. Biophys. Res. Commun. 157. I 117-1123. [2 ] Dobbs. L.G.. Gonzalez. R. and Williams. M.C. (1986l Am. Rev. R~,0ir. Dis. 134, 141-145. [2.~;] Laemmli. U K . (1970) Nature 227. 680-685. [2~,] Bradford. M.M. (19761 Anal. Biochem. 72.2-1.8-254. [3tq P¢~rnerantz. A H., Rudolph. S.A.. Haley, B.E. and Greengard. P. ~19751 Biochemistry, 14. 3858-3862.
Biochimicaet BiophysicaActa 131 I ( 1996~ I 17-123
Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells Lln-Jin P. Zimmerman " Mengshu Wang, June B. Nelson, Felix S. Ekwunife, Lin Liu blstitute fiJr Em'ironmental Medicine. Unicersitv t~ Pennsyh ania Medical Ce,lter. Philadelphia. PA 19104. USA
Revi~ed 12 October 1995:accepted6 No,ember 1995
Abstract Stimulation of secretion from rat alveolar epithelial type il cells by the fl-adrenergic agonist terbutaline activates cAMP-dependem protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type 11 cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within I-3 rain of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppres~ble by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two er,dogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for ~cretion. Keywords: Pneumocytetype 11:Terbutaline: Protein kinase A: Calpain
1. I n t r o d u c t i o n Two phenotypically different epithelial cells, type ! and II, line the pulmonary alveolus [1]. Type n cells synthesize lung surfactant which is stored in lamellar bodiex and secreted by exocytosis [2-4]. Previous studies have shown that the secretory activity of type II cells can be enhanced by various secretagogues such as calcium ionophore, parinergic and ~-adrenergic agonists [5,6]. Stimulation of type II cells, for example, with the ~-adrenergic receptor agonists terbutaline or isoproterenol activates adenylate cyclase via G-protein and results in increased cAMP levels that activate PKA [7,8]. Activated PKA then phosphorylates specific protein(s) presumably involved in the secretory processes [9]. Recently we observed that the same set of secretagogues also activates endogenous calpain in isolated alveolar type It cells [10]. Furthermore, type iI cells pretreated with a cell-permeable calpain inhibitor exhibited substantially depressed ( 4 0 - 5 0 % ) secretory ac• Correspondingauthor. Fax: + I (215) 8980868. 01674889/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0167-4889(95)00181-6
tivities [11]. In this study, therefore, we investigated the effect of calpain activation on protein kinase A and its phosphorylation activity in alveolar type I1 cells stimulated with terbutaline. Calpains (EC 3.4.22.17, calcium-activated neutral proteases) are characterized by their ability, :e perform limited proteolysis of specific proteins including protein kinases [12]. cAMP-dependent protein kinase (PKA) [13.14], calcium-phospholipid-dependent protein kinase (PKC) [ 15,16], and pp60 ~r~ tyrosine protein kinase (TPK) [17] are cleaved by calpain. Proteolytically modified protein kinases are functionally altered and can phosphorylate without cofactors. For example, upon binding cAMP, PKA is activated when tts regulatory subunits dissociate from the enzyme. The regulatory subunits of PKA are, however, susceptible to proteolysis by calpain and can be cleaved into 39 and 17 kDa fragments [13]. The proteolyzed regulatory subunit is then no longer able to reassociate with the catalytic subunits to inhibit them and the enzyme remains activated without cAMP [14]. The regulatory fragments, however, retain their ability to bind cAMP and can be photolabeled
U..J.P. Zimmerm,net ul. / Biochhnicaet Biophysh'aActa 1311 t lqV6) 117-123 with 8-N~-[ ~'P]cAMP. a photoaffinity analog of" cAMP [18,19]. cAMP-binding (35-39 kDa) proteins are produced following the endogenous [I 3] or exogenous [20.21] proteolysis of regulatory subunits from both PKA isozymes. These fragments are apparently formed in vivo since their levels are not reduced following homogenization of tissues in the presence of EDTA or other protease inhibitors [14.22.23]. Age-dependent changes in the amount of endogenously proteolyzed PKA regulatory subunits in mouse lung are also reported [24], In the present study, we investigated whether proteolysis of PKA by calpain occurs and engenders sustained phosphorylation activity in stimulated type II cells. The effect of proteolysis on the stimulus-dependent phosphorylation activity of PKA was examined utilizing the colpain inhibitor 11 (N-acetyl-leucyl-leucyl-methioninal). This inhibitor is known to possess good membrane penetratability and is a potent inhibitor of calpain [11,25,26]. The stimulus-induced proteolysis of PKA was monitored by photoaffinity labeling of the proteolyzed fragment. Endogenous proteins specifically phosphorylated by activated PKA were identified by their responses to calpain or PKA inhibitor.
2. Materials and methods
All culture material was purchased from GIBCO Laboratories (Grand Island. NY), [y-3-~P]ATP (2 mCi/ml: 3 Ci/mmol) and [~z P]orthophosphate (1 mCi/ml) were purchased from Amersham (Arlington Heights, IL). 8-N 3[3_,P]cAMP (8-azidoadenosine-3'.5'-cyclic monophosphate. I mCi/ml; 50 Ci/mmol) and monoclonal anti-actin (C4MAb) were purchased from ICN (lrvine, CA). Calpain inhibitor II (N-acetyl-leucyl-leucyl-methioninal) was purchased from Boehringer Mannheim (Indianapolis, IN). Kemptide (cAMP-dependent protein kinase substrate. LRRASLG) and phosphocellulose units for PKA assay were purchased from Pierce (Rockford, IL). Synthetic cAMPdependent protein kinase inhibitor (rabbit sequence TTWADFILASGRTGRRDAILHD), terbutaline, charcoal (at,,ivated) and other chemicals were from Sigma (St. Louis. MO). 2.1. Isolation of ah'eolar epithelial O'pe H cells and preparation ~f c:vtosol fraction Alveolar type 11 cells were isolated from rat lung, as previously described by Dobbs at al. [27]. Isolated type ii cells were plated at 10 million density in 100 mm cell culture dishes (Costar). After 18-20 h of culture in minimal essential medium (MEM) with 10% fetal calf serum (FCS) and in air/CO, (19:1), the medium containing unattached cells was discarded. Adhering type II cells (3-4 million. 30% plating efficiency) were equilibrated further with fresh MEM without FCS or prcincubated wiih MEM
containing 10 /zM of calpain inhibitor II for 45-60 min before initiation of experiments. Inhibitor treated or untreated cells (3-4 million) were subsequently stimulated with terbutaline (10 ,aM) for 0, 1, 2, 3, and 5 min. Control cells (0 rain) had no additions. At the end of each incubation period, activated charcoal (0.3 m g / m l ) was added and the medium was quickly removed. The cells were wa:,hed twice with ice-cold PBS containing I mM EGTA and then scraped gently. Harvested cells were pelleled by cemrifugation at 1000 rpm for 10 rain. The cell pellet was lysed in 100-150 ,al of ice-cold lysing buffer (40 mM Tris, 150 mM NaCI. 2 mM EGTA, 10 mM Chaps. I mM PMSF, I0 mM benzamidine, 0.1 mM leupeptin. 0.1% aprotinin. 0.1 ,aM pepstatin, 0.Irk NAN,, pH 7.0) for 60 rain and further distrupted by sonication (Ultrosonicator. Inc., setting 7) for 5 s twice. Cytosolic supernate was used for PKA phosphorylation activity determination and photoaffinity labeling. 2.2. Assay for phosphorylation actirity of PKA A reaction mixture (50/J,I) containing cytosolic fraction (30-40 ,ag), MgCI 2 (5 mM), ATP (5 ,aM) and Kemptide (40 ,ag) was used for assay of PKA phosphorylation activity. For the PKA inhibited samples, synthetic PKA inhibitor (10 ,aM) was also included. Alter a 3-min preincubation, the reaction was started by adding [3"~P]ATP (2.5 x 106 cpm). Phosphorylation was terminated after 10 min by the addition of an equal volume of stopping buffer (50 mM Tris, 10 mM NaF, I mM DTr, pH 7.6). The reaction mixture was spotted on a phosphocellulose microcentrifuge unit and centrifuged for 30 s. The adhering sample was washed with 250 /1,1 of phosphoric acid (75 mM) and centrifuged. The process was repeated. The phosphocelluse unit containing the processed sample was transferred into a scintillation vial and counted. 2.3. Assay for endogemms proteins phosphorylated by PKA The reaction mixture contained the cytosolic fraction and MgCI,. with or without synthetic PKA inhibitor (10 p,M). The reaction was initiated by adding [~-'P]ATP. Following a 10-min incubation, phosphorylation was terminated by adding solubilizing buffer. The mixture was then heated at 100°C for 5 min and subjected to SDS-PAGE analysis lbllowed by autoradiography. The radioactivity of selected protein bands stained with Coommassie blue was scanned by AMBIS. 2.4. Metabolic htbeling ~f alceolar type H ceils with H: P]orthophosphate After 1~-20 h of culture in MEM with 10% FCS. type II cells plated at I0 million density in 100 mm cell cuture dishes were washed with phosphate-deficient MEM l\,r 6
U.-,I.P. Zmmu', mm et ,I. / Ri,chimica el Bioph~~i~a.,h ta 1311 ¢ItJt)fil I 17-123 times and then incubated with [~ZP]orthophosphate (10 / x C i / m l ) for 3 h. Labeled cells were either pre-treatcd with 10 /aM calpain inhibitor 11 for 60 min or left untreated. Both types of labeled cells were subsequently stimulated with terbutaline (/.aM) lbr 5 rain. The cells were harvested and solubilized in sample buffer and analyzed by S D S - P A G E and autoradiography for endogenously phosphorylated proteins. 2.5. Photoaffbti~." labeling PKA
~ .,:,: _ ~ o ~ 2::-
~,
~5. :'_ 2co
of the regtdatory sttlmnit o f
aliquot (100 p.l, I m g / m l ) of cytosolic fraction was placed ip a microtiter plate well containing a small bar magnet. Photolabile radioactive c A M P (8-N~-[ ~-~P]cAMP. 1 /aM) was added and incubated for 30 min in the dark at r o o m temperature. Photolysis was carried out using a U V S - 5 4 mineralight l a m p 10 c m from the plate for 15 min at r o o m temperature. An aliquot (50 # l ) o f the photolyzed sample was injected into an equal v o l u m e of 5 ~ T C A for determinat;on o f T C A precipitable radioactivity. Another aliquot (50 /zl) was injected into the stopping buffer (50 mlVl Tris. pH 7.6, 10 m M NaF. 1 m M D T ' I ) for analysis of photolabeled proteins by S D S - P A G E and autoradiography.
~ ~:-
An
2.6. Other procedures S D S - P A G E was carried out according to L a e m m l i [28] using 7.5 or 10,% gel. Protein concentrations were determ i n e d by the m e t h o d o f Bradford [29] using bovine 3'globulin as a standard.
c~a
: 3
7
3
2
a
5
Time (min)
Fig. I. The cllccl ,ff calpain inhibition on the stimulus-induced PKA pho~,phor~,l,,li,m ,cli,.it) in alveolar epithelial type n cells. Cells ',,.ere either pretreated :'.ith calpain inhibitor I1 (N-acetyl-leucyl-leucyi-methioninal. II) #M) lor 45-60 min or left untreated. Both types of cells were then ,timulatcd '.,.ith tcrbutaline ( I0/aM) for 0. I. 2.3.5 rain. Cell lysate (30-40/zg) '.'.a~a,,,a'.ed for PKA acti'.ily ,asing Kemptide (40 #g) as the ,ub-trate v.ith or v.ithout s.'.nthetic PKA inhibitor ( I0 #M) as, de:~,cibedin Sccdon 2. [ '-"P] incorporation {pmol/mg protein) wa:~,plotted a~. lunction of qimulatioll time. ~. Untreated cells: A. assayed in the presence of PKA inhibitor: O. cells pre-treated with calpain inhibitor; z~. assayed in the pre,encc of PKA inhibitor. Values are means±S.E, of 4-6 experimcnt~
A
10
-
97 -
3. R e s u l t s
3.1. Effect o f calpain rm P K A a c t i r # v in ~.'pe II cells in response to stimulation T o investigate the effect o f stimulus-induced calpain activation on the P K A activity in type 11 cells, P K A activities o f the cells pretreated with calpain inhibitor were c o m p a r e d with their untreated counterparts. Phosphorylation activity in the calpain-inhibited cells should represent native P K A activity without proteolytic modification. Thus, cells w e r e preincubated with cell-permeable calpain inhibitor II (N-acetyl-leucyl-leucyl-methioninal. 10 ,ttM) for 4 5 - 6 0 min prior to stimulation by terbutaline for 0. I. 2, 3. and 5 min. The inhibitor concentration and preincubation time were previously d e t e r m i n e d by the inhibition o f [3H]phosphatidylcholine secretion from type II cells prelabeled with [3H]choline [1 I] and by inhibition of proteolysis o f e n d o g e n o u s spectrin in intact type II cells [ 1 0 ] P K A activity o f the cytosol fraction was assayed using PKA-specific substrate (synthetic peptide, Kemptide). Fig. 1 indicates that P K A activity rises to a m a x i m u m within 1 - 2 min and thereafter declines during 3 - 5 min o f stimu-
64
!'14-
4 k 1
2
3
4 1
2
3
4
Fig. 2 Photoatlinit) labeling of PKA regulatoo ~,uhunits in type n cells. Acti'~atcd charcoal treated cell ly:.,ate fi'om type II cells stimulated with terhutaline t 10 /z~.l~ Ibr 0. 2. 3 min were photoaffinity-labeled v,ith ;-;-azido-cAMP ( I p..M) as described in Section 2. A: TCA precipitable radioacti~il.~. Aliquot of photolysed mixture was precipitated by equal ,.olume 5c~- "[CA and counts of the precipitate ~erc plotted as function of ~timulation time. Values sho'.,,n are averages of tv,o experiments. B: Photolabeled prolcm~. Aliquot of photolabeled mixture was analyzed by SDS-PAGE (7.5'~ gel) t'~llowed by autoradiography. Lane I. unstimulated celh,: lane 2. 2 rain: lane 3. 3 rain r.timulated cells: lane 4. calpain inhibited ,:cll~.
I,'.-J.P. ZintmelTnan et al. / Biochimictt et Biophysica Acta 1311 19961 117-123
112 97 -
:.,
:.
~mb~mD .,,imp, ~ 1
2
3
~4111.,,imB ¢lmm,.ml 4
5
6
7
8
Fig. 3. Endogenous proteins phosphorylated by PKA in type II cells stimulated b) terbutaline. Cells v.ere stimulated with terbutaline for O, 1. 2. 3 min. Cytosolic fractions (30 /zg) in lysing bufl~r (see Section 2) were incubated with MgCI. (5 raM) and ATP (5 /.tM). For the PKA-inhibited samples, synthetic PKA inhibitor ~I0 ~M) was also present. Alter 3 min preincubation, the reactkm was stalled by addition of [~"PIATP (2.5×10 ~ cpml. After 10 min incubation, the reaction mixture was directly solubilized in sample buffer and analyzed by SDS-PAGE (7.5cb gel) followed by autoradi[zraphy.Lanes (I-4): 0. 1.2.3 min stimulatnon. I~mes (5-8k O. I. 2. 3 min stimulation and assayed in the presence of PKA inhibitor.
type II cells in response to stimulation, the appearance of this fragment was monitored by photoaffinlty labeling. The fragment was covalently labeled wdh a photolabile analogue of radioactive cAMP. 8-azido-cyclic-[ ~2P]AMP. The fragment binds photolabel with high affinity ( 1 2 5 - 2 0 0 nM) and exhibits the same binding characteristics as c A M P [30]. Photolysis of 8-N-[ 32P]cAMP by UV (245 nm) generates a nitrene group which ~:an react with any amino acid residues within its binding site. Isolated type II cells ( 3 - 4 million/plate) were stimulated with terbutaline (10 ~ M ) for 0. 2 or 3 rain. The cytosolic fractions were then preincubated with 8-N 3[ 3 : p ] c A M P (I g,M) for 30 rain in the dark and subsequently photolyzed using a UVS-54 mineralight lamp for IO rain (see Section 2). Aliquots were withdrawn for the determination of TCA precipitable incorporation of radioactive photolable and for analysis of the photolabeled proteins by S D S - P A G E and autoradiography. Fig. 2A indicates that T C A precipitable radioactivity increased during the first 2 - 3 min of stimulation. Analysis of the photolabeled proteins by S D S - P A G E and autoradiography revealed that in the unstimulated cell lysate, the P K A regulatory subunit type 1 (RI, 49 kDa) alone was heavily photolabeled, while the regulatory sub-
" q 112
lation (n = 6), Protracted stimulation of 3 0 - 6 0 min yielded no delayed additional activity. The initial PKA activity of
unstimulated cells (200 pmol/mg protein) increased 1.52.0 fold (367 pmol/mg), reaching a maximum at 2 min
declining to half (260 pmol/mg) by 3-5 rain. The rise and fall of PKA activation was also observed in the calpain-inhibited cells except that m a x i m u m activity occurred consistently earlier (I min) and was 2 2 - 2 5 % lower than the untreated cells (n = 6). When synthetic PKA inhibitor was included in the assay mixture, stimulus-dependent modulation of phosphorylation activities was absent and was 7 0 - 7 5 % less than the m a x i m u m activity of either untreated or inhibited cells. The data suggest that stimulation of type II cells with terbutaline activates calpaiu and PKA, and that calpain up-regulates maximal P K A activity for 1 0 - 1 5 % and prolongs it relative to calpain-inhibited cells.
66-
~.::
!i!¸m 21-
....
3.2. T k e s t i m u h t s - i n & . ' e d cells
proteolysis o f P K A in ~'pe H
Previous study indicated that trypsin (or an endogenous calcium-dependent protease during purification) cleaved the regulatory subunit of PKA isozyme II and generated a distinctive 39 kDa fragment which is still able to bind c A M P [13]. To confirm the limited proteolysis of P K A in
1
2
3
4
5
6
7
8
Fig. 4. The effect of calpain inhibition on the phosphorylation of endogenous proteins in type II cells stimulated by terbutaline. Cells were either pre-treated with calpain inhibitor 11(10 #M) far 60 min or left untreated. Both type of cells were then stimulated with lerbutaline (IO /.tM) for 0, I, 2.3 rain. Cytosolic fractions t60/,~g) was incubated with phosphorylation reaction mixture and processed as described in Fig. 3 legend. Lanes ( I-4): 0, I. 2, 3 rain stimulation of untreated ceils. Lanes (5-8): 0,1, 2, 3 rain stimulation of calFain-inhlbitedcells.
U *J.P. Zimmenmm et al. / Bio¢himi¢ ,~ ¢'t Itiol,hv~i~, A,t,': / ¢! / ~/"961 I /7-123
3.3. E n d o e e m m s p r o t e i n s p h o s p h o r y l a t e d
Pl10
]I c e l l s in r e s p o n s e to s t i m u l a t i o n
100 ;
T o identify the e n d o g e n o u s proteins phosphorylated by PKA in response to stimulation, the standard P K A assay was carried out without Kemptide. Assays were also performed ~ith and without synthetic P K A inhibitor. T h e samples were subjected to S D S - P A G E and auloradiography. From visual estimates o f the intensity o f phosphor vlated protein hands, two proteins with electrophoretic mobility corresponding to 112 and and 47 k D a were j u d g e d to be the e n d o g e n o u s substrates o f P K A . T h e stimulus-dependent phosphor3,1ation kinetics o f these proteins was ( I ) similar to the activity profile observed in vitro (Fig. 3. lanes I - 4 and Fig. 4. lanes I - 4 ) . (2) abolished when P K A inhibitor was present (Fig. 3. lanes 5 - 8 ) . and (3) modulated when calpain inhibitor was present (Fig. 4. lanes 5 - 8 ) . Furthermore, when e n d o g e n o u s phospho~,lation of 3:P metabolically labeled cells was examined, proteins (104, 138. I I 0 , 84. 73, 53, 47, 40 and 29 kDa) were phosphorylated in response to a 5-min slimulaTitm xsith terbutaline. A m o n g these proteins, p110
6o~
o
1 40 -;i
d
by P K A in O'pe
120
i n,"
121
i
0
P47 18
14 -; Z
_o
:
~ 0
a
Z
6
n' p,
,'.
~-i
=!
i
01
'
66
-
! 1
z
3
Fig. 5. In vivo phosphoo'lation of endogenou,~ proteins in phosphorous-32 labeled alveolar type 11 cells stimulated with terbutaline, lsohacd type II cells were labeled with [~:Plorthophosphate (IO p-Ci/n'd) for 3 h. Labeled cells were either pre-treated with calpain inhibitor 11( 10 ~M) for 60 min or left untreated. Both types of labeled cells were subsequentl? stimulated with terbutaline (10 p,M) for 5 min. Cell~, v.ere harse~ted. solnbilized in sample buffer and analyzed by ~DS-PAGE and autoradiography. The radioactivities in p112 and 1347protein hands '.','erescanned by AMBIS and plotted. (I) Unstimuluted cells. (2) (erbutuline-stimulated cells. (3) cells pre-treated with calpain inhibitor and then stimulated with terbutaline.
unit type II (RII, 56 kDa) and a proteolytic fragment o f R (39 kDa) w e r e only faintly photolabeled. In the stimulated cells, h o w e v e r , all three were intensely photolabeled. In addition, a 45 and 43 k D a doublet and a 33 k D a protein were also labeled (Fig. 2B). A radioscan indicated that photolabeling o f the 39 k D a f r a g m e n t increased 6- to 8-fold in the stimulated cells ( n = 2). T h e results indicate that P K A is subject to proteolysis in type 11 cells in response to stimulation and that proteolysis is complete within 2 - 3 min.
45-
~O
~
47
:~,~
21-
1
1
i
W
~
2
3
4
Fi~. 6. Endogenott~ proteins phosphorylated in type II cells stimulated by terbulatine and l~.,a',ed in the presence of exogenous cAMP. Cell lysate from cell, stimulated with terbutaline for O, I. 2. 3 min was assayed for PKA activity in the presence of cAMP (10 /zM) and analyzed by SDS-PAGE ( 10% gel) followed by autonldiogruphy. Lanes ( I ) 0 rain. (2) I min. (~) 2 min. (4) 3 min.
U..Z P. Zimmcrman ,'t al. / Biochimica el Biophysica Acta 1311 t 1996) I 17-123
and p47 showed 20-25c/~ reduced phosphorylation in calpain-inhibited cells relative to uninhibited cells (Fig. 5). The specificity of the endogenous proteins phosphorylated by PKA was further demonstrated by a PKA assay in which exogenous cAMP (I /.tM) was added. Under this condition, the phospho"vlation of p47 was highly enhanced relative to the other proteins (Fig. 6). Thus. the p47 protein appears to bt: the major target of activated PKA in type II cells in response to stimulation by terbutaline.
4. Discussion Enhanced secretory activity of alveolar epithelial type I1 cells is associated with an increased intracellular cAMP (or calcium) concentration and activation of protein kinase PKA (or PKC). Intracellulal cAMP (or calcium), however, rises only transiently, whereas observable enhanced secretion occurs much later at 1-2 h [5]. How the transient rise in the second messengers and subsequent activation of protein kinases relate to the augmcoted secretory activity of type II cells over longer time periods remains obscure. Furthermore. the cellular proteins phosphorylated by activated protein kinase have not been well characterized an~t their exact role in the secretory activity of type 11 cells is presently unknown. In contrast to neurotransmitier release or platelet activation. stimulated secretion of type I1 cells is a relatively slow and protracted process that may require the sustained elevation of second messenger or signal-transducer activity. It is not known whether the process is effected by a prolonged protein kinase activity or by multiple repetitive activations in time. We examined therefore whether the limited proteolysis of PKA by activated calpain might provide a plausible mechanism for sustained augumentation of secretion. Proteolytically modified PKA is known to remain activated without initial activating cofactor cAMP [141. The results of our photoaffinity labeling experiment indicate that limited proteolysis of PKA indeed takes place in type II cells in response to stimulation. It has been shown that trypsin treatment of purified PKA regulatory subunits R I (49 kDa) and R n (56 kDa) results in 37 and 39 kDa fragments, respectively [211. To our knowledge, a comparable study using calpain has not been done. In our own cell system, however, other protein bands between 49 and 39 kDa are also photolabeled. In addition, the photolabeling of the full-length regulatory subunits (56 and 49 kDa) increases in spite of increased proteolysis in response to stimulation. The reason for enhanced photolabeling of the full-length regulatory subunits is not clear. It has been shown that regulatory subunits exist in either holoenzyme form or dissociated state, and that the relative proportion of each may change, for example, with age [24]. A stochastic change in proportionality in response to stimulation
may favor the dissociated subunit with higher affinity for cAMP and thereby result in increased photolabeling. In parallel with proteolysis, PKA activity appears to be sustained at elevated levels longer than calpain-inhibited cells. The peak activities of both cell groups are nevertheless transient, leveling off after 5 rain of stimulation with no later peaks. Hence. our results suggest that secretory activity of type II cells is sustained by continuous augmented activation rather than multiple repetitive activation by PKA. The proteins p112 and p47 proteins specifically phosphorylated by PKA in response to stimulation are potential signal transducers for type II cell secretion. In view of the highly enhanced phosphorylation of p47 protein in the presence of exogenous cAMP. it appears to be the preferred substrate of PKA. Our preliminary studies by immunoblot with monoclonal antibody anti-actin followed by autoradiography, however, suggest that the p47 protein is not an actin molecule. Under our experimental conditions. actin itself was not phosphorylated in type It cells in response to terbutaline, in contrast to a previous study reporting cAMP-dependent phosphorylation of actin in type II cells [9]. In conclusion, stimulus-induced PKA activity is up-regulated and sustained by calpain. The target of PKA activity appears to he two proteins, p112 and p47. The identity of these proteins remains to be elucidated.
Acknowledgements The authors are grateful to Dr. George A. Hermann for his editorial help and to Mrs. Elaine Primerano for the preparation of the manuscript. This work was supported by U.,~. Public Health Service Grant ROl HL49982 (UJ.Z.).
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