High Expression of ,&Adrenergic Receptor Kinase in Human Peripheral Blood Leukocytes ISOPROTERENOL AND PLATELET ACTIVATING FACTOR CAN INDUCE KINASE TRANSLOCATION*

Receptor phosphorylation is a key step in the process of desensitization of the B-adrenergic and other related receptors. A selective kinase (called B-adrenergic receptor kinase, BARK) has been identified which phosphorylates the agonist-occupied form of the receptor. Recently the bovine BARK cDNA has been cloned and the highest levels of specific mRNA were found in highly innervated tissues. It was proposed that BARK may be primarily active on synaptic receptors. In the present study, the cDNA of human BARK was cloned and sequenced. The sequence was very similar to that of the bovine BARK (the overall amino acid homology was 98%). Very high levels of BARK mRNA and kinase activity were found in peripheral blood leukocytes and in several myeloid and lymphoid leukemia cell lines. Since agonist-induced BARK translocation is considered the first step involved in BARK-mediated homologous desensitization, we screened a number of G-protein-coupled receptor agonists for their ability to induce BARK translocation. In human mononuclear leukocytes, B-AR agonist isoproterenol and platelet-activating factor were able to induce translocation of BARK from cytosol to membrane. After 20 min of exposure to isoproterenol (10 PM), the cytosolic BARK activity decreased to

Exposure of cells containing @-adrenergic receptors (PAR)' to P-adrenergic agonists results in rapid and reversible loss of the receptor-mediated response to subsequent stimulation (homologous desensitization) (1). A number of mechanisms underlying homologous desensitization have been well characterized in vitro. They include receptor phosphorylation, uncoupling from G., and sequestration (1). It is now accepted that receptor phosphorylation is a key step in the process of receptor desensitization (1). A selective kinase (called Padrenergic receptor kinase, PARK) has been identified (2-8) which phosphorylates the agonist-occupied form of the receptor. Receptor phosphorylation by PARK requires an additional cytosolic factor, called B-arrestin (9), to induce complete homologous desensitization. PARK phosphorylates in an agonist-dependent manner not only PAR but also some other G-coupled receptors: the a2-adrenergic receptor (3), muscarinic cholinergic receptors (4), and, to a lesser extent, rhodopsin (2). Additionally, isoproterenol, prostaglandin El and somatostatin (6,10) induce translocation of PARK from cytosol to membrane, suggesting that their receptors are also substrates for PARK phosphorylation. Recently, the bovine PARK cDNA has been cloned ( 5 ) and the highest levels of specific mRNA were found in highly innervated tissues ( 5 ) . It was proposed that BARK may be primarily active on synaptic receptors (1).
In the present study, the cDNA for human PARK was cloned and sequenced. High levels of PARK expression were observed in human peripheral blood leukocytes (PBL), which, together with the ability of isoproterenol and platelet-activating factor (PAF) to induce BARK translocation, suggests a role for PARK in modulating some receptor-mediated immune functions.
PCR reactions were carried out according to Ref. 11 with minor modifications. To obtain the first cDNA strand, 1 pg of poly(A)+ RNA (in a few cases total RNA) from mononuclear leukocytes (MNL) was reverse-transcribed using random hexamers. The cDNA was amplified in 100 p1 of PCR buffer (10 mM Tris-HC1, pH 8.3, 50 mM KCl, 1.5 mM MgC12, 0.01% gelatin), 0.8 pg each of F and R primers, 200 PM each of the dNTPs, 2.5 units of Thermus acquaticus DNA polymerase (Amplitaq, Perkin-Elmer/Cetus). PCR cycles were modified for our cloning strategy. PCR was performed for 36 cycles, with 1 min of denaturation at 94 "C, annealing for 1 min at various temperatures (see below), and 4 min extension at 72 "C. The annealing was done at 42 "C for the first three cycles (low stringency PCR) and increased stepwise to 47 "C (three cycles) and then to 55 "C (30 cycles, high stringency PCR). This protocol drastically reduced background products when compared with annealing performed at 42 "C for all the cycles (not shown). The amplification finished with 10 min at 72 "C to generate blunt-end products. PCR products were subcloned blunt-end in PTZ18R and used for sequencing.
cDNA Library Screening-A human pituitary cDNA library in Xbluemid (cloning site Eeo RI and HindIII; Clontech, Palo Alto, CA), containing 1.5 X lo6 recombinants was screened (12). Two PCR products from bp 1666 to 2067 (F5-R1 in Fig. 1) and bp 1 to 705, labeled by random priming (Amersham International kit), were used as a probe for two independent screenings. Screenings were done under high stringency conditions (12).
DNA Sequeneing-Nucleotide sequence analysis of both strands was performed by the dideoxynucleotide chain termination method, by primer extensions with T7 DNA polymerase (United States Biochemicals or Pharmacia LKB Biotechnology), using as template plasmid DNA prepared according to Maniatis et al. (12). Each clone was sequenced in both directions.
Human Tissue and Cell Sources-Macroscopically normal tissues, obtained from surgically excised samples, were rapidly frozen at -70 "C. Heart samples were papillary muscle from open heart surgery for mitral valve disease; pericardic adipose tissue was from patients undergoing myocardial transplantation for idiopathic congestive cardiomyopathy; lung and liver samples were taken 6-8 cm away from the periphery of tumor tissue, usually in a different lobe. Cultured cells (American Tissue Culture Collection), growing under standard conditions in appropriate media supplemented with 5-10% fetal bovine serum, were harvested directly in guanidinium isothiocyanate.
PBL from healthy volunteers, were fractionated as previously described (13). Briefly, MNL were isolated by Ficoll gradient, and in some cases they were further fractionated into lymphocytes and monocytes by a Percoll gradient. Granulocytes were isolated from erythrocytes by Percoll gradient.
Northern Blot Analysis-Total RNA was isolated by the guanidinium isothiocyanate/cesium chloride method (12). Total RNA (20 pg) was fractionated on a 1% agarose-formaldehyde gel and transferred to a Genescreen Plus membrane (Du Pont-New England Nuclear). The RNA blot was hybridized with a random primed radioactive cDNA fragment (bp 1055-1946) in 50% formamide, 10% dextran sulphate, 1% SDS, 5.8% NaCl, and denatured salmon sperm DNA (100 pg/ml) for 24 h at 42 "C. The blot was washed in 2 X SSC, 1% SDS at GO "C, 0.1 X SSC at room temperature and was subjected to autoradiography overnight at -80 "C. All the results were confirmed on RNA from at least two different individuals.
Preparation of Cytosolic and Membrane Fractions for Assay of BARK-The method used was based on that described previously (5). Briefly, cells or tissue fragments were pelleted by centrifugation (800 X g for 5 min), lysed in cell lysis buffer (10 mM Tris, 5 mM EDTA, 7.5 mM MgCl,, 0.1 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, 5 pg/ml pepstatin, 10 pg/ml benzamidine, at pH 7.4) using a polytron tissue disruptor (Janke and Kundel) at low speed for 1 min on ice. Unbroken cells and cell nuclei were pelleted by centrifugation (800 X g for 5 min) and discarded. The supernatant was then centrifuged at 48,000 X g for 20 min at 4 "C to separate the plasma membrane and cytosolic fraction, the latter was then centrifuged at 150,000 X g for 60 min at 4 "C. The protein content of the resultant supernatant was measured using Bio-Rad protein assay reagent, and its BARK activity was assayed on the same day (cytosolic BARK assay). Membrane preparation was washed once in cell lysis buffer, recentrifuged at 48,000 X g for 15 min at 4 "C, the resultant membrane pellet resuspended in cell lysis buffer, sonicated for 5 s prior to protein determination.
Bouine Rod Outer Segments Preparation-Previous studies demonstrated that rhodopsin contained in the ROS of bovine retina can be phosphorylated by PARK in a light-dependent manner (2), and that it can be used to investigate PARK activity quantitatively (6). Thus, rhodopsin was used in the present study to measure and compare PARK activity of various cell and tissue preparations. ROS were prepared from bovine retina by stepwise sucrose gradient sedimentation using a method described in Ref. 8. Retinal rhodopsin kinase was degraded by treatment with 5 M urea (14).
Phosphorylation Assay-The phosphorylation reaction was based on that previously described in Ref. 6. Briefly, in each reaction, 100

BARK Expression and
Translocation in PBL pg of total cytosolic soluble protein or membrane protein was added to a reaction mixture containing 300 pmol of urea-treated ROS, 50 p M [y-"2P]ATP (2-5 cpm/fmol), 20 mM Tris, 8 mM MgC12, 3 mM EDTA, 5 mM NaF, 12 mM NaCl, 0.07 mM phenylmethylsulfonyl fluoride, 7 pg/ml leupeptin, 3.5 pg/ml pepstatin, 7 pg/ml benzamidine, at pH 7.4 and a total volume of 150 pl. The reaction was carried out at 30 "C in the presence (or absence) of light for 45 min. The reaction was stopped by the addition of 900 pl of an ice-cold solution containing 10 mM Tris, 100 mM NaCl, 10 mM NaF, 2 mM EDTA, followed by centrifugation a t 57,000 X g for 15 min at 4 "C. The resultant pellet was reconstituted in SDS sample buffer and electrophoresed on 10% SDS-polyacrylamide gel.
BARK Translocation Assay-Freshly prepared MNL (2 X lo6 cells/ ml) were incubated in RPMI 1640 with 1% glutamate, 1% penicillin/ streptomycin at 37 "C (pH 7.4) without serum for 30 min. Various agonists were then added at different concentrations and incubated a t 37 "C for the indicated times. At the end of reaction, ice-cold PBS was added, cells were pelleted, and crude BARK was prepared as described.
SDS-Polyacrylamide Gel Electrophoresis-This was carried out using a method previously described (15). Following electrophoresis on a 10% homogeneous slab gel, the gel was stained for protein with Coomassie Blue. Following destaining, the gel was dried and subjected to autoradiography. For a quantitative measurement of BARK activity, two methods were used (1) rhodopsin bands (molecular mass -35 kDa) identified by Coomassie Blue staining, were cut and counted for "P radioactivity; (2) measurement of relative density of rhodopsin bands imprinted on the autoradiographic films by densitometry (RAS, Amersham).
Materials-PCR Amplitaq DNA polymerase was obtained from Perkin-Elmer/Cetus; deoxynucleotides used for PCR were from Pharmacia LKB Biotechnology Inc.; the modifying enzymes end restriction endonucleases were from Bethesda Research Laboratories, from Pharmacia, and from Boehringer Mannheim. The DNA probes for hybridization were prepared with the Amersham random priming kit. The human cDNA library was purchased from Clontech Laboratories Inc. (Palo Alto, CA). The hybridization filters were purchased from Du Pont-New England Nuclear. Tor 32P-labeled dATP and dCTP were purchased from Amersham Corp. PKI (P3294), heparin (H-3125) from porcine intestinal mucosa, vasoactive intestinal peptide, and complement 5a were purchased from Sigma; 1,5-(isoquinolinesulfonyl)-2-methylpiperazine (designated H7) and staurosporin were from Calbiochem; PAF was purchased from Bachem (Bubendorf, Switzerland); BN52021 is a gift of Dr. P. Braquet (Institute Henri Beaufour, Le Plesis-Robensir, France); [32P]ATP was from Du Pont-New England Nuclear. All gel electrophoresis materials were purchased from Bio-Rad.

RESULTS
Cloning and Sequencing of Human PARK-The human PARK cDNA was cloned by PCR, with F and R primers complementary to the bovine cDNA sequence (5). To account for possible PCR errors, most of the sequence was confirmed in clones obtained from at least two different PCR amplifications. Most of these clones were also overlapping, so that the sequence of the oligos used for PCR was confirmed in clones spanning these regions. The 3' region was obtained by screening a human pituitary cDNA library using two PCR products as probes. Two PARK cDNA fragments (approximately 1.3 and 2.8 kb in size) were cloned and sequenced from bp 153 to the first 30 bp in the untranslated region (Fig. 1). The human PARK cDNA sequence obtained displayed a very high similarity with the bovine cDNA (93% identity). The overall amino acid identity was 98% (Fig. 1).
PARK mRNA Tissue Distribution-To examine mRNA distribution in human tissues and cells, the 891-bp fragment F4-R2 was used as a probe for Northern blot analysis. Similar to what was observed in bovine tissues, we found a major mRNA species 4 kb in size with, in some tissues, an additional species of 2.4 kb (Fig. 2). As previously reported for bovine, heart and lung showed moderate to low levels of PARK mRNA, while it was not detectable in liver and adipose tissue (Fig. 2). By contrast, PARK-specific transcript was very abundant in MNL (approximately 4-5-fold higher than in heart, as quantitated by densitometric analysis of the autoradiogram). Such an unexpected finding suggested a preferential expression of PARK in immunocompetent cells. This idea was further supported by Northern blot analysis of several different cultured cell types (Fig. 3). We compared 16 different human cell types: four lymphoid leukemia cell lines (HPB-ALL, U937, MOLT4, Jurkat), one myeloid leukemia (HL60), one erythroid leukemia (K562), two hepatomas (Hep G2 and SK-HEP-l), and one each embryonic lung fibroblasts (MRC5), lung carcinoma (A549), neuroblastoma (IMR-32), breast adenocarcinoma (MCF7), ovarian carcinoma (SW626), osteosarcoma (0 143), endothelial cells (4th passage), smooth muscle cells (25th-27th passage, SMC). A detectable PARK mRNA was found in all the cell types, and the highest levels of expression were observed in all the lymphoid and myeloid cell lines (Fig. 3). In these blots, the level of mRNA in PBL, used as internal control, was within the range of lymphomyeloid cell lines (Fig.  3). Direct comparison of PARK expression between human PBL and brain (these are the two tissues with the highest levels of PARK mRNA) was not possible. In fact, several experiments with fragments from surgically excised brain samples never yielded RNA of quality good enough to be used for Northern blot analysis. To overcome this problem, PARK expression was compared in mRNA from bovine PBL and brain (Fig. 4). The level of PARK expression in bovine PBL was found to be slightly higher than in the brain (Fig. 4).
Bovine brain RNA used in these experiments was obtained from frontal cortex.
PARK Activity Tissue Distribution-In order to study whether the high levels of specific mRNA present in MNL are indeed translated into high concentrations of PARK in these cells, a biochemical assay was used to measure the PARK activity in different cells and tissues. According to previous studies (6), bovine rhodopsin was used as specific substrate for phosphorylation. Under our experimental conditions, in the presence of soluble proteins from MNL, rhodopsin was highly phosphorylated (Fig. 5). The kinase activity calculated from four different individuals was 10. nM, not shown) and protein kinase A inhibitor PKI (1 p M ) (Fig. 5). As previously shown (16), the less specific protein kinase C inhibitor H7 (10-100 p~) , at the highest concentration used, slightly inhibited rhodopsin phosphorylation (Fig.  5, lane h). No such phosphorylation was observed when ROSor MNL-soluble proteins were incubated separately under identical conditions (Fig. 5). These biochemical features repeat those previously described for bovine brain PARK (7,16), suggesting that our experimental conditions are suitable for measuring PARK activity. Comparison was then made of the relative expression of PARK activity in MNL, SMC, Jurkat cells, and liver and lung tissues (Fig. 6). The highest activity was found in MNL and Jurkat cells with little or no detectable activity in SMC and liver and lung tissues (Fig. 6). This pattern of PARK activity closely resembles the relative amount of mRNA expression in these cells and tissues.
Agonist-induced PARK Translocation-Since agonist-induced PARK translocation is considered the first step involved in PARK-mediated homologous desensitization (lo), we screened a number of G-protein coupled receptor agonists for their ability to induce PARK translocation in human MNL. Isoproterenol (10 p~) , the PAR agonist, induced translocation of cytosolic PARK, after 20 min of exposure to isoproterenol, the cytosolic PARK activity decreased to 60.7 +. 6.3% ( n = 4) of control (Fig. 7), while membrane-associated PARK activity increased to 170% ( n = 2) (not shown). This effect of isoproterenol has previously been observed in S49 and DDT, MF-2 smooth muscle cells (6,lO). Although this is a novel observation so far as the human immune cell is concerned, we searched for other receptors that were also able to induce PARK translocation. For this purpose, VIP, C5a, and PAF were chosen for having direct effects or their receptors present on MNL. Five and twenty minutes were chosen as incubation times for inducing translocation. Neither VIP (0.5 p~) nor C5a (100 nM) had any observable effect (data not shown), while PAF was able to induce translocation in a time-and dose-dependent manner (Figs. 8-10 6. Relative tissue and cell distribution of BARK activity. Soluble proteins (100 pg) from cells or tissues were added to 300 pmol of urea-treated ROS in the presence (+) or absence (-) of 10 pg/ml heparin and incubated for 45 min a t 30 "C in phosphorylation buffer. Samples were then centrifuged a t 57,000 X g for 15 min and the pellet was resuspended in SDS buffer and electrophoresed on a 10% SDS-polyacrylamide gel. Autoradiography of dried gels was for 1-2 h a t -70 "C. Two independent experiments are shown. In the left gel, BARK activity was compared in MNL (lanes a and b ) , liver (lanes  c and d ) , and lung tissue (lunes e and f ) . In the right gel BARK activity was compared in MNL (lanes g and h), SMC (lanes i and j ) , and Jurkat cells (lune k). Data represent a t least two separate experiments. Freshly prepared MNL were exposed to isoproterenol for 0, 5, and 20 min, and then ice-cold PRS was added and the cells pelleted. Cell pellets were resuspended in cell lysis buffer and were sonificated, and unbroken cells and dehris removed by centrifugation. The supernatant was centrifuged a t 48,000 X g for 20 min to separate the memhrane and t.he cytosolic fractions. The latter was then centrifuged a t 150,000 X g for 60 min a t 4 "C; the resultant supernatant contained the crude [fARK preparation, which was added to the phosphorylat.ion assay a t a quantity of 100 pg of total protein and processed as described.

PARK Expression and Translocation
[jARK activity is expressed as 9;) of control (100%). Data represent two to five experiments. Insert shows ROS hands on autoradiographic film from one representative experiment. Freshly prepared MNL were exposed to PAF for the indicated times and then ice-cold I'RS was added and the cells were pelleted. Crude BARK preparation was done as descrihed in Fig. 7. PARK activity is expressed as o/o of control (100%). Data represent two to five experiments. Inserts show ROS hands on autoradiographic film from two independent time courses. f 5.4% ( n = 7) and 52.0% (n = 2) of control, respectively (Fig. 8), with concomitant increases in membrane bound PARK activity to 214 and 171% of control at the same time points (n = 2) (Fig. 9). In addition, 20 PM of the PAF receptor antagonist BN52021 (17) blocked the effects of 1 PM PAF (Fig. 10).

DISCUSSION
The cDNA of human BARK was cloned and sequenced. The sequence is very similar to that of the bovine BARK (the overall amino acid homology is 98%) showing very high interspecies conservation. This feature is common to several other kinases and is likely to be due to the relevant functions of such enzymes. A 891-bp fragment of cDNA was used for Northern blot analysis of mRNA from different tissues and cells (Figs. 2 and 3). The most striking finding of this study was that BARK mRNA is expressed in noninnervated cells, with very high levels of expression in PBL. In parallel, a very high BARK activity was found in MNL (Figs. 5 and 6), suggesting that these high mRNA levels are indeed translated in PRI, Freshly prepared MNI, were exposed t o I'AF for the indicated times, and then ice-cold P H S was added and the cells were pelleted. Following sonification in cell lysis huffer. unhroken cells and cell tlehris removed. memhrane preparation was ohtained hy centrifugation at 48.000 X fl for 20 rnin a t 4 "C. After washing in cell lysis hrlffer. 100 pg of total memhrane protein was added t o the phosphorylation reaction as descrihed. /fARK activity is expressed as of cnntrol ( 1 0 0 ). Data represent two experiments. Insrrl shows ROS bands on autoradiographic film from one representative experiment. The presence of such a high level of /?ARK expression in PBL suggests a functional role for this kinase in these cells.
Indeed, in MNL, PAR agonist isoproterenol and F' AF were ahle to induce translocation of pARK from cytosol to the membrane, which appears to be the first step in homologous desensitization (10). That isoproterenol was ahle to induce translocation of PARK came as no surprise since PAR has been shown to be a suhstrate for pARK (lo), and its activation induced SARK translocation, alheit in different cell types (6,10). Since catecholamines have heen shown to have numerous modulatory effects on immune cells (18), our ohservation suggests that pARK is directly involved in the catecholaminergic regulation of immune functions.
PAF is a phospholipid with diverse potent effects, ranging from regulation of platelets. lvmphoc.ytes, monoqytes, gmnu-locytes, to pathological responses such as in asthma and allergy (for review see Ref. 19). PAF receptor has recently been cloned and shown to be a member of the G-proteincoupled receptor family, with multiple serine and threonine residues in the C-terminal region as potential phosphorylation sites (20), and PAF has been shown to induce homologous desensitization (21,22). Protein kinase C has been implicated to be only partially responsible for PAF-induced homologous desensitization (23), which is in line with the presence of protein kinase C phosphorylation site sequence motifs in the C-terminal region of PAF receptor. The present observation of PAF-induced BARK translocation in MNL suggests a role for BARK as well. The situation here may be analogous to that of PAR desensitization, i.e. BARK-mediated phosphorylation is responsive to receptor activation at high agonist concentrations (24), since we observed BARK translocation only at PAF concentrations of 10-100 nM or more. Thus, the notion that PAF receptor and BAR in MNL act as substrates for BARK, backed by the high levels of BARK expression in these cells, directly supports the claim of a role for PARK in human immune functions.
In the light of the present findings, the working hypothesis that BARK may be active only at the synapses level (1, 51, need to be expanded. We suggest that, in addition to the synaptic receptors, BARK may be a potent modulator of at least some receptor-mediated immune functions. In this regard, the recent cloning of some peptide hormone receptors (25,26), thrombin receptor (27), chemotactic receptor for formyl peptide fMet-Leu-Phe (28), interleukin-8 receptor (30, 31) (all seven-membrane spanning domain receptors) provide further evidence that the number of cellular functions which are mediated by G-coupled receptors is far larger than previously expected. These receptors appear to be good candidates for BARK regulation as they do contain C-terminal tails rich in serine and threonine residues, potential phosphorylation sites for BARK (29). Additionally, at least some of them (i.e. thrombin and formyl peptide met-Leu-Phe receptors) became desensitized after exposure to their respective agonists (homologous desensitization) (27,32). Therefore, it is possible that PARK may serve to regulate a wider spectrum of immune cell receptors, in addition to PAF and @adrenergic receptors.
An additional point of interest raised by the present observation of PAF-induced BARK translocation concerns the nature of BARK receptor substrates; PAF-receptor activation is shown to be coupled to the activation of phospholipase C , while all the known receptor substrates for PARK identified so far are coupled to adenylate cyclase, namely PAR, muscarinic acetylcholine receptor, cY2-adrenergic receptor, somatostatin, and prostaglandin El receptors. While it has been suggested that BARK phosphorylates only the adenylate cyclase-related G-protein-coupled receptors, no BARK translocation factor has yet been identified. Thus, it remains possible that the nature of the second messenger system may not necessarily reflect the ability of one receptor to induce translocation of BARK.
The analysis of BARK transcript from different human cells and tissues raises several points of potential interest (Fig. 3). First, a detectable amount of BARK transcript was found in all of the tested cells, whereas it was not observed in several tissues or organs (including liver, muscle, and adipose tissue (Ref. 5 and present study)). Second, PARK transcript, although undetectable in normal liver, was quite abundant in two differentiated hepatoma cell lines (Hep G2 and SK-Hepl). Third, as expected for nonlymphomyeloid cells, the highest levels of expression were found in neuroblastoma cells (IMR-32). Finally, PARK transcript, highly abundant in all of the lymphoid and myeloid cell lines tested, was poorly expressed in K562 cells, which are highly undifferentiated blasts sharing some characteristics of the erythroid lineage. Several questions raised by these observations are presently under investigation in our laboratory.
This study also demonstrates that MNL contain high levels of BARK activity which can be easily detected by a phosphorylation assay using bovine ROS as a substrate. This assay thus provides a useful tool for further investigation of BARK activity in PBL in physiological and pathological conditions in humans.
During the preparation of this manuscript, two papers were published which are related to the present work (33,34). The first shows the sequence of human PARK cDNA as obtained by screening a human retinal library. The coding region of the two sequences (Ref. 33 and present paper) differed only in bps 631, 1265, 1394, and 1716. The second paper (34) reported on the cloning of a second bovine subtype of BARK (referred to as BARK 2). Based on the relative homology with the two bovine BARK subtypes (5, 34), the present sequence must be considered as the human form of the BARK 1. Additionally, since PARK 2 was five times less efficient than PARK 1 in phosphorylating rhodopsin (34), we are confident that our kinase assay, based on rhodopsin phosphorylation, mostly measures PARK 1 activity.
In conclusion, we provide here the first evidence for a high level of human PARK expression and activity in PBL. With the support of the data on isoproterenol and PAF induced BARK translocation in MNL, we suggest that PARK plays a role in modulating at least some receptor-mediated immune functions.