(Received for publication, September 20, 1995; and in revised form, January 2, 1996)
From the
Tyrosine kinase activity, a determinant of Src homology domain interactions, has a prominent effect on cellular localization and catalysis by 5-lipoxygenase. Six separate inhibitors of tyrosine kinase each inhibited 5(S)-hydroxyeicosatetraenoic acid formation by HL-60 cells stimulated with calcium ionophore, in the presence or absence of exogenous arachidonic acid substrate, indicating that they modulated cellular 5-lipoxygenase activity. The tyrosine kinase inhibitors also blocked the translocation of 5-lipoxygenase from cytosol to membranes during cellular activation, consistent with their effects on its catalytic activity. These results fit a model which postulates that Src homology domain interactions are a molecular determinant of the processes which coordinate the subcellular localization and functions of 5-lipoxygenase. In addition, we demonstrate that activated leukocytes contain two molecularly distinct forms of 5-lipoxygenase: a phosphorylated form and a nonphosphorylated form. In activated HL-60 cells the pool of phosphorylated 5-lipoxygenase accumulates in the nuclear fraction, not with the membrane or cytosolic fractions. The amount of phosphorylated 5-lipoxygenase is a small fraction of the total. Overall, equilibrium reactions involving the nuclear localizing sequence, the proline-rich SH3 binding motif, and the phosphorylation state of 5-lipoxygenase may each influence its partnership with other cellular proteins and any novel functions derived from such partnerships.
5-Lipoxygenase (5-LO) ()catalyzes the formation of
leukotriene (LT) mediators of inflammation(1, 2) . In
resting neutrophils, 5-LO is usually confined, in an inactive state, in
the cytosol. Agonist stimulation initiates the translocation of 5-LO
from the cytosol to cell membranes where it can associate with an
activating protein, termed
FLAP(3, 4, 5, 6, 7, 8, 9, 10, 11) .
Translocation and interaction with FLAP are determinants of 5-LO
activity in the simplified model of cellular LT formation(7) .
This model explains the mechanism of action of certain
anti-inflammatory agents(12, 13, 14) ;
however, it is imprecise in five respects. First, there is no evidence
for a direct 5-LO-FLAP interaction; all data supporting their
interaction are correlative (12, 13, 14) .
Second, stabilization of 5-LO by phospholipids, in vitro,
fully accounts for effects originally attributed to FLAP (15, 16) . Third, in cells lacking FLAP, 5-LO still
translocates from the cytosol to the membrane, implying that 5-LO can
bind to membrane components other than FLAP(7) . Fourth, in
certain leukocytes, 5-LO occurs in the cell membrane or nucleus in the
resting state(8, 11, 17) . Thus, in
nonactivated cells, cytosolic localization of 5-LO is not a general
rule, and membrane localization of 5-LO does not necessarily correspond
with FLAP interaction(18) . Fifth, LT formation by receptor
mediated events, such as IgE cross-linking in mast cells, depends on an
unidentified signal, not a Ca
threshold(19, 20) . Thus, the oversimplified
model of cellular LT formation prompted us to investigate molecular
determinants governing the redistribution or activation of 5-LO. We
recently reported that 5-LO associates with other proteins via Src
homology domain interactions, specifically via interactions with SH3
domains. We now report that cellular tyrosine kinase activity, a
determinant of SH2 domain interactions, has a prominent, previously
overlooked, influence on activity and cellular localization of 5-LO and
that activated leukocytes contain two molecularly distinct forms of
5-LO, a phosphorylated form, and a nonphosphorylated form.
In certain experiments we determined the subcellular distribution of 5-LO among the cytosolic, plasma membrane, and the nuclear soluble and particulate fractions of resting and A23187-stimulated HL-60 cells. Differentiated HL-60 cells were disrupted by nitrogen cavitation. The cytosol, membrane, and nuclei were isolated, and 5-LO was visualized by immunoblotting as described(11) .
HL-60 cells were differentiated into granulocytes in the presence of
[P]orthophosphate. Six flasks, each with 20 ml,
2
10
cells/ml in phosphate-free RPMI 1640 media,
10%, v/v, fetal bovine serum, 1.25% Me
SO, 2 mML-glutamine, and penicillin/streptomycin were incubated
for 12 h in 5% CO
. After confirming cell viability at 12 h,
[
P]orthophosphate (2.5 µCi/ml) was added to
each flask. After 6 days HL-60 cells were isolated by centrifugation at
100
g, washed once with phosphate-free RPMI medium, 10
mM HEPES, 1 mM CaCl
, pH 7.4, and
resuspended in this buffer (2
10
cell/ml)
containing [
P]orthophosphate (100 µCi).
Cells were stimulated with 2.5 µM A23187 or 2 µM PMA for 10 min, 37 °C, then quenched with 5 mM EDTA.
Cells were centrifuged (100
g) and washed 2
2
ml of phosphate free buffer. The cell pellet was suspended in 500
µl of lysis buffer (phosphate-buffered saline, 1 mM sodium
orthovanadate, 1% Triton X-100, 0.1% sodium deoxycholate, 0.01% SDS, 5
mM EGTA, 5 mM EDTA, 50 mM sodium
pyrophosphate, and 5 mM benzamidine), frozen on dry ice, and
thawed. After four freeze-thaw cycles, the sample was centrifuged at
12,000
g on a microcentrifuge for 10 min. In this
case, cell lysate supernatant fraction contains both microsomal and
cytosolic 5-LO; the cell lysate pellet contains genomic DNA and any
nuclear associated proteins. The cell lysate supernatant fraction,
containing the microsomal and cytosolic 5-LO, was removed and
transferred to a separate microcentrifuge tube containing 15 µl of
anti-5-LO antiserum (1/67 final dilution). The cell lysate pellet,
containing genomic DNA, was washed using minimal shear stress, with 3
1 ml with 0.05 M Tris-HCl, pH 7.5, 10 mM MgCl
, then suspended forcefully in 500 µl of
buffer containing DNase I (50 µl, 0.2 µg/µl, 89
units/µg). After digestion of DNA for 1 h at 25 °C, the lysate
pellet was centrifuged at 12,000
g
10 min on a
microcentrifuge. The supernatant fraction (500 µl), containing
proteins originally associated with intact DNA, was mixed with 2
lysis buffer (500 µl) and 15 µl of anti-5-LO antiserum.
The digested DNA fraction (cell lysate pellet) and the soluble
microsome/cytosol fraction (cell lysate supernatant) were incubated for
3 h at 25 °C to permit immune complex formation between anti-5-LO
antibody and any 5-LO in the samples. Protein A-agarose was then added
(40 µl, 1:1, w/v, in 1
lysis buffer); the sample was
incubated for 1 h at 25 °C and then the resin containing
5-LO
anti-5-LO complexes was isolated by centrifugation for 30 s
at 12,000
g on a microcentrifuge. The agarose resin
was washed 3
1 ml with lysis buffer, suspended in 20 µl of
2.5
electrophoresis buffer, and boiled for 5 min. The entire
immunoprecipitate was fractionated by SDS-PAGE on a 10% polyacrylamide
gel with a 4% stacking gel. The supernatant fraction from the protein
A-agarose immunoprecipitate was also fractionated by SDS-PAGE.
Following electrophoresis, gels were fixed for 30 min with 10% acetic
acid, 30% ethanol, then in 0.5% glycerol/water prior to drying.
Proteins containing
PO
were detected by
exposing gels to Kodak XAR-5 film 10-20 days at -80 °C.
Prior to immunoprecipitation with the anti-5-LO antiserum all samples
were ``cleared'' of proteins which interacted nonspecifically
with normal rabbit serum (1/67 dilution) and protein A-agarose. The
tracking dye front, which contained a majority of the free
P background, was removed from the gel prior to exposure.
However, the exposure time necessary to detect the immunoprecipitated
78-kDa 5-LO band still produced substantial background from
P-labeled oligomeric DNA fragments in the section of the
gels from 39 kDa to the tracking front. For 10-20 day exposures
we were unable to eliminate this background by extensively washing the
protein A-agarose immunoprecipitate without jeopardizing the 5-LO/LO-32
binding to the resin.
Figure 1:
Effect
of tyrosine kinase inhibitors on cellular 5-lipoxygenase. A,
dose-dependent inhibition of 5-HETE formation by HL-60 cells stimulated
with A23187. B, dose-dependent inhibition of 5-HETE formation
by HL-60 cells stimulated with A23187 in the presence of 20 µM arachidonic acid. Curves were fit to the data using the program
Graphpad to estimate inhibitor potency. The rank order of inhibitor
potency was: 2,5-DHC > genistein > herbimycin > tyrphostin
lavendustin > compound 5 in each panel. Values represent the
mean ± S.E., n = 4-6
experiments.
Figure 2: Effect of tyrosine kinase inhibitors on purified 5-lipoxygenase. Dose-dependent inhibition of 5-HETE formation by purified 5-LO incubated with 20 µM arachidonic acid. Curves were fit to the data using the program Graphpad to estimate inhibitor potency. The rank order of inhibitor potency with the isolated enzyme was: lavendustin > 2,5-DHC > tyrphostin > compound 5 > genistein > herbimycin.
Figure 3:
Effect of tyrosine kinase inhibitors on
cellular 5-lipoxygenase translocation. Neutrophils (2 10
cells/ml) were incubated with tyrosine kinase inhibitors or 0.4
µM MK-886 for 2 min prior to stimulation with 1 µM A23187 for 10 min to initiate translocation 5-LO from cytosol to
membranes. 5-LO in the 100,000
g supernatant (cytosol)
and the 100,000
g pellet (membranes) was monitored
immunochemically with a 5-LO-specific antiserum. The amount of
membrane-associated enzyme is depicted directly below the amount of
cytosolic enzyme. In all panels lane 1 = resting
neutrophils, lane 2 = stimulated neutrophils, lanes
3-5 = stimulated neutrophils plus tyrosine kinase
inhibitor, lane 6 = 0.4 µM MK-886. Panel A = 2,5-DHC; panel B =
herbimycin; panel C = tyrphostin 25; panel D = compound 5; panel E = lavendustin; panel F = genistein. Each lane contains equal amounts
of protein (10-20 µg/lane).
We verified by
immunoblotting that 5-LO in resting HL-60 cells was most abundant (80%
of total) in the cytosolic pool; however, 5-LO also occurred in the
soluble nuclear protein fraction. This is analogous to results reported
for rodent cells(11, 17) . When HL-60 cells were
activated with A23187, 5-LO redistributed from the cytosolic pool to
the 100,000 g nuclear particulate fraction. Enzymatic
assays for 5-LO activity supported the immunoblotting experiments. For
instance, we detected 5-LO activity in the cytosol and nuclear soluble
fraction of resting cells and the 5-LO activity redistributed to the
nuclear particulate fraction in A23187-stimulated cells (data not
shown).
Figure 4:
Incorporation of PO
into cellular 5-LO.
PO
-labeled 5-LO
occurred in the immunoprecipitate of the DNA fraction from HL-60 cells
stimulated with A23187 (lane 2), but was less abundant in the
DNA fraction of control cells (lane 1) or cells stimulated
with PMA (lane 3). The exposures necessary to detect the
immunoprecipitated 78-kDa 5-5-LO band produced substantial
background from genomic DNA fragments. The darkened portion of
the gel, near 35 kDa, is a portion of this P
-labeled
nucleic acid background, not a protein
band.
When purified 5-LO isolated
from human leukocytes, or recombinant 5-LO, was incubated with
[-
P]ATP, then analyzed by SDS-PAGE and
autoradiography, an indistinct band of
PO
co-migrated with the purified protein at 78 kDa. The signal/noise
ratio was always low and barely distinguishable above the background.
This indicates that tight association of
[
-
P]ATP with a putative ATP binding site in
5-LO does not account for the radiolabeled phosphoprotein observed in
the 5-LO immunoprecipitate depicted in Fig. 4. Phosphorylation
of 5-LO by a cellular kinase best explains the data.
Neither the serine/threonine kinases, MAP and Cdc-2, nor the tyrosine kinase, Lyn, catalyzed phosphorylation of isolated 5-LO in vitro under the conditions described. Autophosphorylation of the kinases occurred, indicating that they were catalytically active.
Compartmentalization and functions of 5-LO depend on
processes other than binding to
FLAP(8, 9, 10, 11) . Modulation of
5-LO translocation and catalysis by tyrosine kinase inhibitors fits a
model which postulates that Src homology domain interactions are a
molecular determinant of these processes(34) . There are two
types of Src homology domains(35) . The SH2 domain, consisting
of approximately 100 amino acid residues, binds to tyrosine residues
phosphorylated by tyrosine kinase. Intermolecular binding between SH2
domains and tyrosine phosphate residues initiates the cellular
redistribution of proteins. The SH3 domain, consisting of approximately
60 amino acid residues, binds to proline-rich regions of amino acid
residues(36) . Notably, 5-LO contains a proline-rich motif that
interacts with the SH3 domain of certain signaling
proteins(34) . Signaling proteins, adaptor proteins, or Src
kinases usually contain both SH2 and SH3 domains, enabling them to
assemble multimeric complexes(35) . Our model suggests how
cellular tyrosine kinase and Src homology domain interactions could
coordinate 5-LO compartmentalization and activity (Fig. 5). For
instance, cytosolic 5-LO, via its proline-rich motif, could equilibrate
with a protein containing an SH3 domain(34) . If this protein
also contained an SH2 domain then the 5-LOSH3
SH2 complex,
via its unoccupied SH2 domain, could equilibrate with tyrosine
phosphate residues on proteins distributed in the cytoskeleton,
membrane, or nucleus. The order in which these reactions occur and the
specific proteins involved requires further investigation.
Figure 5:
Proposed model of depicting molecular
determinants of 5-LO: protein interactions. Free 5-LO in the cytosol
can equilibrate among three pools: (i) a pool defined by interaction of
5-LO with SH3SH2 domain proteins, (ii) a pool defined by the
redistribution of the 5-LOSH3
SH2 complex among tyrosine
phosphate (pY) ligands for the SH2 domain, (iii) a pool
defined by interaction of 5-LO with FLAP.
As
depicted in our model, 5-LO, itself, need not be phosphorylated to
influence its distribution and activity. However, we demonstrate,
directly and for the first time that cells can phosphorylate 5-LO. Our
results contradict Rouzer and Kargman who concluded that
phosphorylation of 5-LO does not occur(3) . We attribute the
differences to adequate labeling of cells with carrier-free
[P]orthophosphate and to examination of the
nuclear fraction from cellular homogenates. Investigators often
neglected the nuclear fraction of cells until experiments showed that
5-LO can translocate and accumulate at the nuclear
membrane(8, 9, 10, 11, 17, 37) .
Although novel, the significance of our observation is uncertain.
Phosphorylation of 5-LO may influence its translocation and function.
For instance, the nuclear localizing sequence, the proline-rich SH3
binding motif, the FLAP-binding domain, plus the phosphorylation state
of 5-LO may determine its partnership with other cellular proteins and
whether it has functions other than lipid mediator catalysis. It is
notable that phosphorylated 5-LO is most abundant in the nuclear
fraction, not the cytosol or membrane fraction of cells activated by
A23187; however, this is a small fraction of the total 5-LO. The amount
of phosphorylated 5-LO and its specific activity were insufficient to
determine if it contains phosphoserine, phosphotyrosine, or
phosphothreonine. 5-LO contains several consensus sequences for
tyrosine phosphorylation typified by DYI, at residues 20-22 and
residues 94-96 and EYL at residues 538-540. 5-LO also
contains three tyrosines clustered at residues 660-662, about
12-15 residues from the C terminus. These sequences are unique to
5-LO, not 12-LO and 15-LO (38) . If 5-LO were tyrosine
phosphorylated it might interact directly with SH2 domains. 5-LO also
contains a consensus sequence, YLSP, at residues 662-665 for
phosphorylation by MAP kinase (39) and a consensus sequence,
RKSS, at residue 521-524, for phosphorylation by S6
kinase(40) . There is also a consensus sequence for Cdc-2
(p34
) kinase, SPDR at residues 664-667 in 5-LO.
All three mammalian lipoxygenases contain consensus sequences for
phosphorylation by protein kinase A(41) . These are: RRCT at
residues 247-250 in 5-LO, RRST at residues 242-245 in
12-LO, and RRSA at residues 242-245 in 15-LO. However,
lavendustin inhibits protein kinase A and C poorly.
To fortify certain conclusions we used a panel of chemically distinct agents which inhibit receptor and Src tyrosine kinase activity by different mechanisms. Herbimycin, a benzoquinone, reacts irreversibly with sulfhydryl groups near the active site on Src kinase(22, 23) . Lavendustin competitively inhibits both ATP and substrate binding; (26) tyrphostin-25 competitively inhibits only substrate binding; genistein, an isoflavone, competitively inhibits only ATP binding to receptor tyrosine kinases, typified by the epidermal growth factor receptor. 2,5-DHC inhibits substrate binding competitively and ATP binding noncompetitively. These agents can affect cellular 5-LO catalysis, independent of any tyrosine-kinase mediated processes; however, this effect alone would not account for two observations. First, the rank order of potency for inhibition of cellular 5-LO and isolated 5-LO reverses for certain inhibitors, e.g. lavendustin, compound 5, and tyrphostin (Table 1). Second, inhibitors of 5-LO catalysis or redox status do not inhibit translocation or interaction with FLAP. The respective effects of 2,5-DHC, herbimycin, tyrphostin, genistein, and lavendustin on 5-LO translocation correspond best with their effects on cellular tyrosine kinase activity. This conforms with precedents showing that tyrosine kinase coordinates the activation and localization of other enzymes involved in lipid mediator biosynthesis and leukocyte activation(42, 43, 44, 45) . It is possible that all six tyrosine kinase inhibitors act like MK-886 and block 5-LO-FLAP interactions. However, the divergence among their chemical structures and their relative potencies as inhibitors of translocation, which approximate their potencies for inhibition of cellular tyrosine kinase, make this unlikely. We are presently unable to test this without access to radiolabeled FLAP ligands.
Although the effect of the tyrosine kinase inhibitors on isolated 5-LO may seem ``nonspecific'' it is predictable and interpretable from two perspectives. First, lavendustin, compound 5, genistein, and 2,5-DHC each inhibit ATP binding to tyrosine kinase. 5-LO, like tyrosine kinase, requires ATP as a co-factor for catalysis(15) . The sequence and locale of the ATP binding site on 5-LO are uncertain; however, our results are compatible with a similarity between the ATP binding site on tyrosine kinases and the site on 5-LO. Second, all of these inhibitors are ``redox''-sensitive. Certain redox sensitive agents inhibit 5-LO catalysis via effects on the oxidation state of the hemoprotein(46) . We stress, again, that inhibitors of 5-LO catalysis or redox status rarely modulate its translocation or interaction with FLAP. Thus, the effect of tyrosine kinase inhibitors on 5-LO translocation was not predictable and is most compatible with a process involving tyrosine kinase activity.
In summary: (i) tyrosine kinase inhibitors uniformly inhibited the activity of both isolated and cellular 5-LO, (ii) tyrosine kinase inhibitors uniformly inhibited the translocation of 5-LO in leukocytes stimulated with A23187, and (iii) 5-LO occurs as a phosphoprotein in the nuclear fraction of activated leukocytes. These results add to the notion that 5-LO may have a novel role within the nucleus(11, 17, 34) .