1. No category

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1996 87: 202-210
Phosphatidylinositol-3-kinase activity is required for the anti-igmediated growth inhibition of a human B-lymphoma cell line
M Beckwith, RG Fenton, IM Katona and DL Longo
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Phosphatidylinositol-3-Kinase Activity Is Required for the Anti-IgMediated Growth Inhibition of a Human B-Lymphoma Cell Line
By Margaret Beckwith, Robert G. Fenton, lldy M. Katona, and Dan L. Longo
Stimulation of B lymphocytes through the Ig receptor initimannin, can completely abrogateanti-lg-medmted growth
ates acascade of biochemicalchanges, which can ultimately
inhibitionwithout affecting tyrosine kinase induction
or prolead to either activation and growth, or cell-cycle arrest and tein kinase C (PKC) activation.
Treatment of intact cells with
celldeath.Oneof
the critical events that occurs in both
Wortmannin results in an irreversible decrease in anti-lgcases isthe activation of tyrosinekinases, and the resulting
induced P13K activity, suggesting that the effect of Wortphosphorylationof a variety of proteins on tyrosine residues.
mannin on anti-lg-mediated growth inhibition is caused by
In this report we identify one ofthe substrates of phosphory- its inactivation ofP13K activity. Taken together, these data
lation as the 85-kD subunit ofthe enzyme phosphatidylinosi- show that activation of P13K is a critical component of the
tol-3 kinase
(P13K),
andshow
that both anti-lgM and
anti-lg-initiated signaling cascadethat leads to growth inhianti-lgD stimulation results in anincrease
in the antibition of human B lymphoma cells.
phosphotyrosine-precipitable P13K activity.Furthermore,
This is a US government work. There areno restrictions on
we show that the potent and specific inhibitor of P13K. Wortits use.
T
HE Ig RECEPTOR ON B lymphocytes has been shown
to consist of a complex of proteins similar to those
identified as members of the T-cell receptor
The
IgM and IgD heavy and light chains are associated with a
heterodimer consisting of Iga and Igp or Igy,"' products
of the mb-l and B29 genes, re~pectively.~.~
These small
phosphoproteins serve to link the Ig receptor with several
src-family tyrosine kinases that are activated after receptor
ligati~n.~"'
This results in the phosphorylation of several
proteins on tyrosine,'*.l3including the ~ 2 1 GTPase-activat"~
ing protein ( ~ 2 1 "GAP),I4
~
phospholipase C y l (PLCyl)lS
and y2 (PLC ~ 2 ) , ' VAV,"
~ , ' ~ phosphatidylinositol-3 kinase
(P13K),19 in addition to the mb-l and B29 gene products.2a22
The phosphorylation of proteins on tyrosine is a key regulatory process inmany different pathways shown tobe involved in activation and proliferation of B lymphocytes,2'-25
andthe functional role of these phosphorylated signaling
proteins is under intense investigation.
One of these proteins that has recently been the focus of
many studies in a variety of cell types is P13K.26This enzyme
is comprised of a 110-kD (p1 10) catalytic subunit and an 85-
From the Biological Carcinogenesis and Development Program,
Science Applications International CorporatiodFrederick; and Biological Response Mod$ers Program, the Division of Cancer Treatment, National Cancer Institute, Frederick CancerResearch and
Development Center, Frederick;and Departments of Pediatrics and
Medicine, Uniformed ServicesUniversity ofthe Health Sciences,
Bethesda. MD.
Submitted May 22, 1995; accepted August 22, 1995.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human Services,
or the Department of Defense, nor does mention of trade names,
commercial products, or organizations imply endorsement by the
US Goverment.
Address reprint requests to Margaret Beckwith, PhD, NCIFCRDC, Bldg 567, Room 204, Frederick, MD 21702.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solelv to
indicate this fact.
This is a US government work. There are no restrictions on its use.
0006-4971/96/8701-0033$0.00/0
202
kD (p85) regulatory
and it phosphorylates inositol
lipids on the D-3 hydroxyl position." Activation of PI3K
has been shown to occur after receptor ligation inmany
different systems, including growth factor receptors containing intrinsic tyrosine kinase activit~,'~-~'
as well as receptors associated with src-like kinase^.'^^'"^^ For example, the
85-kD subunit of PJ3K has been shown to associate with the
SH3 domaid6 of the lyn tyrosine kinase in B cells,19 to be
phosphorylated on tyrosine after stimulation through surface
Ig," and to bind to purified Ig-aGST fusion proteins, via an
associated src-like k i n a ~ eLigation
.~
of CD19 on B cells has
also been shown to recruit active PDK to the CD19 comp l e ~ . 'In
~ addition, in T lymphocytes, stimulation through
CD28 results in association of CD28 with p85, phosphorylation of p85 on tyrosine, and activation of P13K.3s,'9
Several approaches have been used to determine the functional importance of PI3K in receptor-mediated biologic responses. By mutating or deleting the residues in the plateletderived growth factor (PDGF) receptor critical for PI3K p85
binding,"" or by overexpressing a mutant p85 to which the
110 subunit cannot bind:' investigators have demonstrated
a requirement for PI3K activity in PDGF-mediated receptor
internali~ation,~'membrane ruffling:'
and mit~genesis.~'
Recently, the functional significance of PI3K has also been
demonstrated using inhibitors of PBK activity. One compound that has been shown to be a potent and specificinhibitor of PI3K in vivo and in vitro is the fungal metabolite
Wortmannin.4'." Yano et a14' showed that Wortmannin
blocks the IgE-mediated histamine release from RBL-2H3
cells through a direct interaction of this compound with the
1 10 subunit of PI3K. Taken together, these results strongly
suggest that PI3K is a critical, and shared, signaling component of many different receptor-activated pathways.
We have been investigating anti-Ig-mediated growth inhibition of a human B-lymphoma cell line that expresses
endogenous IgM and IgD, and is negative for Epstein-Barr
virus (EBV). We have previously shown that tyrosine phosphorylation appears to be critical for growth inhibition induced by anti-IgM antibodies bound to acrylamide beads.45
In this report we demonstrate that both anti-IgM and antiIgD induce tyrosine phosphorylation of the regulatory, p85,
subunit of PI3K, and stimulation of enzyme activity. Further-
Blood, Vol 87, No 1 (January l), 1996: pp 202-210
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203
P13K ACTIVITY IN HUMAN B LYMPHOMA CELLS
more, w e show that Wortmannin can specifically block antiIg-mediated stimulation of PDK activity, and anti-Ig-induced growth inhibition, with no effect on the activation of
tyrosine kinases or protein kinase C (PKC). These results
show that PI3K activity is a critical element in the Ig-activated signaling pathway leading togrowth inhibition in these
cells.
MATERIALS AND METHODS
Cell lines and culture conditions. The RL cell line was grown
from the ascites of a patient with diffuse large cell lymphoma. It is
an IgM’, IgD+, B-cell line that has been shown by Southern blot
analysis to be EBV genome negative, and has been described previously.46The cells are passed twice weekly in RPM1 1640 (CellGro;
Mediatech, Washington, DC), containing 2 mmoVL L-glutamine,
1,OOO U/mL penicillin, and 100 pg/mL streptomycin, and 10% fetal
calf serum (FCS), all obtained from GIBCO (Grand Island, NY),
with no other growth factors added.
Reagents. Acrylamide-conjugatedrabbit-antihumanIgM (RcrIgM)
(p-chain specific) and control rabbit y-globulin (Ryy)-acrylamide
beads were purchased from Bio-Rad (Richmond, CA). Murine antihuman IgM monoclonal antibody (MoAb) (DA4-4) was obtained
from the American Type Culture Collection (ATCC) (Rockville,
MD). Murine antihuman IgD MoAb (6IA6.2) and control murine
MoAb of the IgG2a subclass (UPC-10) were a kind gift of Dr Fred
Finkelman (Uniformed Services University of the Health Sciences
[USUHS], Bethesda, MD). Anti-IgM (MaIgM), anti-IgD (MaIgD),
and UPC-l0 conjugated to high-molecular-weight dextran as previously described4’ were a kind gift of Dr Andrew Lees (USUHS,
Bethesda, MD). Rabbit antisera specific for PI3K was obtained from
Upstate Biotechnology, Inc ([UBI], Lake Placid, NY). Wortmannin
was obtained originally from Dr Allen Oliff (Merck & CO,Inc, West
Point, PA), and then purchased from Sigma Chemical CO (St Louis,
MO). Wortmannin was dissolved in dimethyl sulfoxide (DMSO) to
2 or 10 m o m stocks, stored at -20°C. and diluted in media immediately before use.
Proliferation assays. Lymphoma cells were split 18 to 24 hours
before assays were performed. RL cells were resuspended in culture
medium to a concentration of 1 X l@/mL, and 100 pL was plated
in 96-well, flat-bottomed, microtiter plates already containing 100
pL of appropriately diluted reagents. In some experiments RL cells
were pretreated with Wortmannin before adding to the assays. One
microcurie of ’H-thymidine/well (6.7 Cdmmol; New England Nuclear Research Products, Boston, MA) was added for the final 8 to
18 hours of a 72-hour culture. Cultures were harvested onto glass
fiber filters with a PhD Cell Harvesting system (Cambridge Technology, Inc, Cambridge, MA), and isotope uptake was assessed by liquid
scintillation on a beta counter (LKB/Wallac, Inc, Gaithersburg, MD).
Immunoprecipitation and Western blotting. RL cells were incubated with Ryy or RaIgM beads, or MaIgM or MaIgDdextran for
various lengths of time, pelleted, and lysed in buffer containing 20
m o m Tris, 2 m o m EDTA, 1% NP40, 137 m o w NaCI, and 10%
glycerol, with aprotinin (2 pg/mL), phenylmethylsulfonyl fluoride
(PMSF) (100 pmom), leupeptin (5 pg/mL), and Na,V04 (1 mol/
L) added immediately before lysing. Lysates were incubated on
ice for 15to30 minutes, and cleared by spinning 12,OOOg in a
microcentrifuge for 30 minutes at 4°C. Protein concentrations were
determined by BCA protein assay (Pierce Chemical CO, Rockford,
IL), and 50 pg/lane was used for straight immunoblotting. For immunoprecipitations, 200 pg to 500 pg (approximately 2 to 5 X lo6 cell
equivalents, respectively) of lysate was added to Eppendorf tubes
containing Protein A Sepharose (Pharmacia LKB Biotech, Piscataway, NJ) and 10 pL of appropriate antisera. Tubes were rotated 2
hours to overnight, and pellets were washed three times in lysis
buffer, and one time in 10 m o m Tris containing . l % NP40. Electrophoresis sample buffer was added to the washed pellets, samples
were boiled for 10 minutes, and run by 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 8% mini-gels
(Novex, Encinitas, CA). Gels were transfered to Hybond-ECL nitrocellulose (Amersham Corp, Arlington Heights, IL), and blocked for
1 hour in Tris-buffered saline containing 0.1% Tween-20 (TBST),
5% powdered milk, and 5% normal goat serum (TBSThIBiNGS).
Primary antibodies were added, and blots were incubated for 2 hours
at room temperature. After washing (three times, 15 minutes) in
TBST, horseradish peroxidase-linked secondary antibodies were
added to the blots in TBSThIBiNGS. Blots were then washed (three
times, 15 minutes) in TBST, and developed using enhanced chemiluminescent (ECL) kits from Amersham.
PI3 kinase assays. Assays for PI3K activity were performed by
U cells were cultured in the absence
the method of Kaplan et al.@I
or presence of Wortmannin, and then immunoprecipitates were prepared from control or anti-Ig-stimulated RL cells exactly as described above. They were washed three times in lysis buffer, and
three times in 10 m o m Tris, pH 7.4. Wortmannin was added to the
pellet in various concentrations, and samples were incubated at room
temperature for 30minutes. Ten microliters of sonicated PI substrate
(Avanti Polar Lipids, Inc, Alabaster, AL) was added to each sample
and samples were incubated for 10 minutes on ice. Forty microliters
of kinase buffer containing 30 m o m HEPES pH 7.4, 30 m o m
MgCI2,50 pmoVL adenosine triphosphate (ATP), and 200 p m o m
adenosine was added, followed by 10 pCi y-”P ATP (New England
Nuclear). Samples were incubated for 20 minutes at room temperature, and the reaction was ended with 100 pL 1 m o m HCI. Phospholipids were extracted with 200 pL chlorofondmethanol (1: 1). Equal
volume aliquots from the bottom chloroform layer were spotted onto
thin-layer chromatography plates (MCB reagents; Merck & CO,Inc,
Gibbstown, NJ), and developed in chlorofondmethanol/dH20/ammonium hydroxide (45:35:7.5:2.5). Plates were dried briefly,and
placed with x-ray film (Eastman Kodak, Rochester, NY) for 1 to 7
days at -70°C.
RESULTS
Phosphorylation of P13K p85 on tyrosine occurs after
anti-Ig stimulation of RL cells. The RL cell line can be
induced to undergo activation-induced growth inhibition in
the presence of
phorbol
anti-IgD
(data
not shown),
or anti-IgM45antibodies. The latter response was shown to
depend on induction of tyrosine phosphorylation
after receptor ligati~n.~’
To better understand signaling pathways that
might be involved in anti-Ig-induced growth inhibition, we
were interested in identifying specific substrates of tyrosine
phosphorylation. We initially examined the ability of antiIg toinducephosphorylationof
PDK p85. Lysates from
RL cells ( 2 to 5 X lo6 cell-equivalents) treated with Ryy-,
RaIgM-beads, UPC-dextran, or MaIgM-dextran were immunoprecipitated with antibodies
specific for PDK (p85),
electrophoresed, transferred to nitrocellulose, and analyzed
for phosphotyrosine-containing proteins. As shown in Fig
1A, anti-IgM-beads induced phosphorylation of the regulatory subunit (p85) of PDK. In addition, we observed phosphorylation of a protein a t 110 M), which was not reactive
with an antibody specific for the1 IO-kD catalytic PI3K subunit (data not shown), and an unidentified protein at greater
than 200 kD. Probing the same blot with an anti-p85 anti-
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BECKWITH ET AL
204
IP:
IP:
Anti-PI3K
I
0
10'
20'
--n
M M
'
I
M M
CB pB CB pB
A
-
47
-
33
-
0
10'
20'
n
n
-
'
IP: Anti-PI3K
I
CB pB CD pD
CB pB CB pB
-.-
C
B
11084
Anti-PI3K
p85
+p85
+p85
Fig 1. Phosphorylationof P13K by anti-lgM beads or by anti-IgM conjugated to high-molecular-weightdextran. RL cells were treated with
R y y beads (CB),anti-lgM-beads (CB), UPC-dextran (CD), or anti-lgM-dextran (pD) for 10 or 20 minutes (A and B), or l 0 minutes (C). The 0
timepoint is a media only control (M).Three hundred micrograms (a2to 5 x 10' cells) oflysate per sample was immunoprecipitatedovernight
at 4°C as described, and pellets were washed, run on 8% gels, and transferred to nitrocellulose. Blots were then probed with anti-PTyr (A and
C). The blot shown in (A) was stripped and reprobed with anti-P13K(p85) (B). These data are representativeof at least three experiments, with
the exception of the anti-lgM-dextran, which was performed twice.
body (Fig 1B) demonstrated that equal amounts of PI3K
pendent kinases," at concentrations ranging from 5.5 pmol/
(p85) were immunoprecipitated in every lane. In addition,
L (c-src) to 270 pmoVL (PLC). When 1 pmol/L Wortmannin
when we analyzed lysates from RL cells treated for 10 minwas included in the PI3K assay (Fig 2A, lanes 5 through
Utes with high concentrations of anti-IgM-dextran (5 p&!
8), the anti-PTyr-precipitable PI3K activity from anti-lgtreated RL cells was completely abolished. Furthermore, a
mL) (Fig IC), we were also able to detect phosphorylation
dose response of Wortmannin in the in vitro assay showed
of PI3K ( ~ 8 5 )and
. associated proteins. Similar results were
a significant reduction of PI3K activity at 1 nmol/L (Fig 2B,
obtained with anti-IgD-dextran (data not shown). In similar
lane 3), with a complete inhibition observed at 10 nmoVL
experiments using anti-IgM-beads, we were able to measure
(lane 4). These inhibitory concentrations of Wortmannin are
tyrosine phosphorylation of VAV, GAP, and PLC-y (data
consistent with published reports demonstrating the specinot shown), all of which have been implicated in signaling
ficity of Wortmannin for PI3K at nanomolar concentrathrough the Ig re~eptor.'~""
tions?'." Furthermore, the specificity of the in vitro kinase
Anti-IgM and anti-IgD induce increases in anti-PTyrprecipitable PI3K activiol in RL cells, and this activity is
assay for PI3K activity was determined by including adenosine in the assay buffer as an inhibitor of PI4K;* and by
inhibited in vitro by thePI3K inhibitor, Wortmannin. We
showing that NP40 also inhibits the precipitated PI3K activnext performed in vitro PI3K assays to measure directly the
ity (data not shown). These data show that both anti-IgM
ability of anti-Ig to stimulate PI3K activity in RL cells. RL
and anti-IgD stimulate PI3K activity in RL cells, and that
cells were stimulated for 10 minutes with Ryy-beads,
this activity can be specifically inhibited in vitro by the PI3K
RdgM-beads, UPC-dextran, or anti-IgD-dextran. Lysates
inhibitor Wortmannin.
were immunoprecipitated overnight with anti-PTyr, and precipitates were analyzed for PI3K activity. As previously
In vivo treatment of RL cells with nanomolar concentrashown by Gold et aljSfor normal B lymphocytes, we demontions of Wortmannin reverses the anti-Ig-induced growth
arrest, and inhibits PI3K activio. As previously menstrate here (Fig 2A) that anti-Ig stimulation of RL cells with
tioned, PI3K activation has been shown to occur after ligaanti-IgM-beads (lane 2), or anti-IgD-dextran (lane 4) retion
of
different growth factor
the
and
sulted in an increase in anti-PTyr-precipitable PI3K activity many
p85 subunit has been shown to associate with cell-surface
over control lysates (lanes 1 and 3). Wenextwanted
to
proteins involved in costimulation of T cells'R.'') and B cells.37
measure the effect of the PI3K inhibitor Wortmannin in this
To determine if PI3K activity is a critical functional composystem. Wortmannin was isolated as an antifungal compound
from the bacterial culture broth of Penicillium ~ortmannii.~' nent of anti-Ig-induced growth inhibition, we used the PI3K
inhibitor, Wortmannin. RL cells were treated with various
It has been shown to specifically inhibit PI3K at nanomolar
concentrations of Wortmannin for 30minutes at 37°C before
concentration^^^." by binding to the catalytic, 1 I O - k D subthe addition of control or anti-IgM-beads. In two representaunit?' It has been shown to have no effect on many other
intracellular signaling enzymes, including PI4K," PKC,4'."
tive experiments shown in Fig 3, it is clear that Wortmannin
was able to significantly reduce the anti-Ig-mediated inhibic-src-like tyrosine kinases?"
phosphoinositide-specific
tion of RL cells at a concentration of 5 nmol/L, and comphospholipase C," c-AMP-, c-GMP-, and calmodulin-de-
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P13KACTIVITY
205
IN HUMAN B LYMPHOMA CELLS
2
1
3
5
4
7
8
8
-
A
PI-3P
Kinase Assay:
Pretreatment:
Fig 2. Anti-lgMandanti-lgD
Activator:
induce
anti-PTyr-precipitable
P13K activity in RL cells, and this
activity is inhibitedin vitro with
the P13K inhibitor, Wortmannin.
(A) RL cells were stimulatedwith
Ryy-beads (CBI, RalgM-beads
(FBI, UPC-dextran (CD),or anti&dextran (&D) (Activator). Lysates wereprecipitated
overnight
with
anti-PTyr, and
assayed for P13K activity as described in Materials and Methods, in the absence or presence
of 1p m o l l L Wortmannin (Kinase
Assay). Position of phosphatidylinositol 3-phosphate indiis
cated. (B)
Dose
response of
Wortrnannin inhibitionof in vitro
P13K assay. RL cells were stimulated with Ryy-beads (CB) or
RalgM-beads (FBI. Lysates were
immunoprecipitated
overnight
with anti-PTyr, and assayed for
P13K activity in the absence or
presence of various concentraWM in Assay
tions of Wortmannin, as indicated under the figure. Position WM Pretreat:
ofphosphatidylinositol 3-phosActivator:
phate is indicated.
1
2
3
4
5
6
B
-
PI-3P
(PM):
0
CB
pletely abrogate the response at concentrations of 10 to 50
nmol/L. These results show that Wortmannin interferes with
signal transduction through surface Ig, presumably via its
effect on PI3K, and thus abrogates growth arrest.
Wenext assayed the in vivo effect of Wortmannin on
PI3K precipitated from anti-lg-treated RL cells. RL cells
were treated with various concentrations of Wortmannin for
30 minutes at 37"C, followed by stimulation with anti-Ig
beads for I O minutes.As shown in Fig 4A, whenPI3K
activity was measured in anti-PTyr immunoprecipitates from
RL cells treated in vivo with concentrations of Wortmannin
ranging from 0.5 to 5,000 nmolL, inhibition of enzyme
activity was observed. In Fig 4B, anti-PTyr immunoprecipitates from the same lysates were run on a 10% SDS-PAGE
mini-gel, andblottedwithanti-P13Kp85MoAbtoshow
that equivalent amounts of phosphorylated PI3K p85 were
brought down in every lane where samples had been treated
with anti-Ig. A phosphotyrosine immunoblot (Fig 4C) of the
lysates fromwhich
the immunoprecipitates weremade
shows that there was no effect of Wortmannin at anyconcentration on anti-lg-induced tyrosine phosphorylation, and immunoblots of these lystates with anti-PI3K p85 (Fig 4D)
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
206
BECKWITH ET AL
40
50
35
30
45
n
I
0
25
x
5
20
40
/+'
U
15
35
10
5
30
I
0
3.0
10.0
100.0
1000.0
I/+
I
b
.O
0.5
50.0
5.0
[Wortmannin] nM
shows that there were equal amounts of PBK in each sample.
These data, taken together, strongly suggest that Wortmannin
interferes with anti-Ig-induced signaling and growth arrest
of RL cells via its inhibitory effect on PI3K, thus implicating
PI3K as a critical functional component of this response.
Wortmannin does not interfere with anti-lg-induced tyrosine kinase activation or stimulation of PKC. We next
wished to monitor the specificity of Wortmannin in our system, especially because our initial experiments were performed with a relatively high concentration (5 pmol/L). We
further assessed the effect of Wortmannin on tyrosine kinase
activation after anti-Ig stimulation of RL cells (see also Fig
4C). RL cells were cultured overnight in the absence or
presence of 5 prnoVL Wortmannin. Cells were then treated
for 10 minutes with media, Ryy-beads, or RaIgM-beads in
the continued absence or presence of 5 pmol/L Wortmannin.
Lysates were either tested directly in immunoblots for induction of phosphotyrosyl-containing proteins (Fig SA), or were
first precipitated with an anti-PI3K p85 antibody before
probing with an anti-PTyr antibody (Fig 5B). In neither case
did we see any reduction in phosphorylation in lysates made
from Wortmannin-treated cells, demonstrating that Wortmannin is not interfering with anti-Ig-induced tyrosine kinase activation or tyosine phosphorylation of P13K p85.
We previously showed that RL cells were growth inhibited
in the presence of phorbol esters, and this inhibition could
be partially reversed by PKC inhibitors,% suggesting that
activation of PKC was occumng. To test the effect of Wortmannin on PKC activation, RL cells were incubated overnight in the absence or presence of 5 pmoVL Wortmannin,
followed by the addition of various concentrations of DMSO
or PMA, and fresh Wortmannin. 3H-thymidine uptake was
measured 72 hours later. As shown in Fig 6, although Wortmannin was able to completely reverse inhibition induced
by anti-Ig-beads, 5 pmoW Wortmannin had no effect on
the PMA-induced growth inhibition of RL cells at any concentration of PMA tested. These data support previous invest i g a t i o n ~demonstrating
~~
that Wortmannin is not interfering
with PKC activation. Taken together, these data show that
Fig 3. In vivo treatment ofRL celhwith Wortmannin completely abrogates enti-lgM-inducedgrowth
arrest. RL cells were pretreated 30 minutes in the
pmence of various concentrations of Wortmannin
in microtiter wells, followedthe
byaddition of media
(A), control beads(0).
or RaIgM-beads ( 0 ) .Cultures
harvested
at were
72 hours, with 'H-thymidine
added
for
the last 18 hours.
The
results
of
two -Derate experiments are shown, and these are representative of four experiments.
in our system, 5 pmol/L Wortmannin has no effect on PKC
or protein tyrosine kinase activation, and therefore support
other reports43." of the relative specificity of Wortmannin
for PI3K.
DISCUSSION
Our previous res~lts,4~
as well as other s t ~ d i e s ? demon~-~~
strated a critical role for tyrosine phosphorylation in Igmediated signal transduction resulting both in proliferation
and growth arrest of B lymphocytes. In this report, we show
that both anti-IgM and anti-IgD result in tyrosine phosphorylation and activation of P13 kinase after receptor stimulation,
and that PI3Kactivity is critical for anti-Ig-mediated growth
inhibition.
It has become clear in the past few years that the membrane kinase PDK is an important signaling molecule shared
by many different receptor complexes. In the case of receptors containing intrinsic tyrosine kinase activity, association
with p85 occurs via p85 SH2 domain binding to p85-specific
Y-X-X-M motifs within the kinase insert d ~ m a i n . * ~For
*~'.~
receptors that lack intrinsic kinase activity, the association
involves binding of p85 to SH3 domains of receptor-associated
In B lymphocytes, Gold et aiJ5 showed
an increase in PDK activity after stimulation with anti-IgM
or anti-IgD. Yamanishi et a l l 9 also showed an anti-Ig-mediated increase in PI3K associated with the lyn tyrosine kinase.
Enzyme activation was shown by Pleiman et a136to occur
via binding of p85 to the SH3 domains of lyn or fyn, which
can themselves bind via SH2 domains to the Ig-associated
signaling molecule Ig-a.'.' Similarly, we show here that antiIgM or anti-IgD stimulation of a human B-lymphoma cell
line results in phosphorylation of PI3K p85 on tyrosine, and
enzyme activation (Figs 2 and3). In preliminary experiments, we have also shown an association of p85 with the
lyn kinase in anti-Ig-activated cells (not shown).
The biologic significance of PI3K activation is unclear in
many systems. Most of the data demonstrating a functional
role for PDK in cellular responses to external stimuli have
been obtained from investigations of PDGF-induced re-
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P13KACTIVITY IN HUMAN B LYMPHOMA CELLS
207
A
1
3
2
4
5
6
8
7
7 -
-
P1 -3P
WM Pretreat (nM):
Activator:
0
M
0
CB
B
1 2 3 4 5 6 7 8
2009768 -
Fig 4. In vivo treatment ofRL
cells with Wortmannin inhibits
anti-PTyr-precipitable P13K activity.(A)
RL cells were pretreated
for
30 minutes with
various concentrations of Wortmannin, followed by stimulation
with control beads (CB)or antiIgM-beads (pB) for
10 minutes at
37'C. Lysates were immunoprecipitated overnight at 4°C with
anti-PTyr MoAb and assayed for
P13K activity as described in Materials and Methods. As a positive control for P13K activity, lysates from anti-lgM-treated RL
cells wereimmunoprecipitated
with anti-PI3K p85 (data not
shown). (B) Lysates fromthe
above experiment were immunoprecipitated with anti-PTyr as
described, run
on
10% SDSPAGE mini-gels, transferred t o
nitrocellulose, andblotswere
probed with anti-P13K MoAb. (C
and D) Lysates from the above
1096
experimentwererunon
SDS-PAGE mini-gels and immunoblots were probed with antiPTyr IC) or anti-PI3K p85 (D) antibodies.
43 -
m-
"
"
"
-
p85
, ' ./
C
200 97 68-
-
43
D
sponses. Fantl et alJoshowed that PI3K p85binds specifically
to two phosphorylated tyrosine residues withinthe kinase
insert domain of the PDGF receptor, and that mutation of
these sites abrogates both the binding of p85, and the mitogenic activity of PDGF. Using similar approaches, Wennstrom et a14' and Joly et al" demonstrated a critical role
for p85 binding in PDGF-initiated membrane ruffling and
receptor internalization and trafficking in the endocytic pathway, respectively. In a T-cell system, investigators have also
recently demonstrated that mutation of the cytoplasmic Y-XX-M domain of the costimulatory molecule, CD28, abolishes
anti-CD28-stimulated binding and activation ofPI3K. as
well as interleukin-2 production."'
Recent studies using specific inhibitors of PI3K activity
1 2 3 4 5 6 7 8
200 97 68-
-
-
.
"
C
"
"
-
p85
43 -
have confirmed and extended these r e s ~ l t s ~ ' ~One
~ " of
~~'~~~'
these inhibitors, Wortmannin, has been shown to selectively
inhibit PI3K activity, without affecting P14K,PKC, W K .
and several other intracellular signaling
In addition to the mutational analysis discussed above,
Wennstrom et a14' showed that Wortmannin could abolish
PDGF-stimulated membrane ruffling. In addition, using
Wortmannin and another PI3K inhibitor, LY294002, other
investigators have shown thatPI3K activation. is required
for insulin or PDGF activation of pp70ShK,.5"andinsulinstimulated DNA synthesis and glucose transporter function."
Finally, in a very detailed analysis of Wortmannin effects,
Yano et alJ demonstrated a role for PI3K in IgE-mediated
histamine and leukotriene release fromtheratbasophilic
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
BECKWITH
208
IP: Anti-P13K
Pretreatment:
- - + - +
Pretreatment:
Activator:
M CB
CB
Activator:
CB
pB pB
- - + +
pB CB pB
"
A
200 97
...
.
-
-
97
p85
68 -
29 -
-
68
-
43
-
43
leukemia cell line, RBL-2H3. In these studies, Wortmannin
treatment of intact cells resulted in irreversible inhibition of
PI3K activation at nanomolar concentrations, probably via
binding of the drug to the pi IO catalytic subunit:' In this
report we have shown that activation of PI3K also appears
to play a critical role in signaling through the antigen receptors on a human B-lymphoma cell line. Stimulation with
anti-IgM or anti-IgD results in the tyrosine phosphorylation
of PI3K p85, activation of PI3K, and growth inhibition (not
shown for anti-IgD). Treatment of the cells with Wortmannin
completely abrogated both growth arrest (Fig 3 ) and PI3K
activity (Fig 4), without affecting p85 phosphorylation (Fig
S). Complete abolition of the anti-Ig-induced growth arrest
and induction of PI3K activity occurred at nanomolar concentrations of Wortmannin (Figs 3 and 4). consistent with
the observed in vitro inhibition of PI3K activity by 1 to 10
nmol/L Wortmannin (Fig 2). Furthermore, consistent with
other results on the specificity of Wortmannin for PI3K. in
our system Wortmanninhadno effect on overall anti-lginduced tyrosine kinase activation (Figs 4 and S), or PMA-
Fig 6. Wortmannin does not interfere with PMAinduced growth inhibition of RL cells. RL cells were
pretreated
overnight
the
in
absence (open symbols)
or presence (closed symbols) of 5 pmol/L Wortmannin. Cells werespun down, andplated in microtiter
wells containing various concentrations of DMSO
(0,W or PMA (A,Al (A), or anti-lgM-beads ( V , + ) (B)
in the continued absence or presence of 5 pmollL
Wortmannin. Cultures were incubated for72 hours,
with 'H-thymidine added for the last 18 hours. The
results are representative of two separate experiments.
AL
Fig 5. Wortmannin does not
interfere with anti-lgM-induced
tyrosine phosphorylation. RL
cells were pretreated overnight
in the absence (-1 or presence
(+) of5pmol/L
Wortmannin.
Cells were then stimulatedwith
media only (MI, Ryy-beads (CB),
or RnlgM-beads (pB) for 10 minutes at 37"C, and lysed with icecold lysing buffer. Lysates were
run directly on mini-gels before
being transferred to nitrocellulose (A), or were immunoprecipitatedovernight with anti-PI3K
p85 antisera before blotting (B).
Blots were probedwith an antiPTyrMoAb, followedby HPOGaMlg and ECL detection. Each
panel is representative of three
separate experiments.
induced PKC activation (Fig 6). even at S pmol/L concentrations. Although these results clearly show that P13K activity
is decreased by Wortmannin in our system, itis possible
that this is not a direct effect, and that Wortmannin is working through another, as yet unidentified, substrate.
Taken together, these studies point to PI3K as a critical
component of signaling pathways through a diverse array of
receptors, in different cell types. Furthermore, it seems to
play a role in a variety of cellular responses ranging from
cytoskeletal changes to receptor internalization and trafficking to inhibition or stimulation of cellular proliferation. The
precise mechanism bywhich PI3K exerts these effects is
unclear. It has been shown thatone of the major endproducts
of PI3K activity, PI-3,4,P2, can activate the zeta isoform of
PKC."It has also been suggested that PI3K may exert its
cytoskeletal function through activation of a small GTPbinding protein, R ~ c . There
~'
is also homology between the
catalytic subunit ofP13Kand a yeast protein, Vps34p, involved in protein delivery to the yeast vacuole,5' againimplicating PI3K in receptor traffickingandprotein
sorting.
[L
-
-
8
40
-
0.0
0.1
1 .o
[PMA]ng/ml
10.0
0.0
5.0
50.0
[Beads]pg/ml
500.0
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
P13K ACTIVITY IN HUMAN B LYMPHOMA CELLS
Therefore, in our system, PI3K could be interacting with a
downstream signaling component that leads to growth inhibition, or alternatively, it could be involved in the internalization of surface Ig after ligation, which may be required for
growth arrest to occur. Experiments are underway to examine these possibilities, and preliminary results suggest that
PI3K may be linked to stimulation of G, in our system (data
not shown).
In summary, we have shown here that ligation of either
IgM or IgD on the surface of a human B-lymphoma cell line
results in tyrosine phosphorylation of PDK p85, enzyme
activation, and growth arrest. The PI3K inhibitor Wortmannin specifically interferes with PI3K activity, and abolishes
anti-Ig-induced growth arrest, thus showing that PDK plays
a critical role in Ig-mediated signaling. Further studies are
needed to define the precise function of PI3K in B-cell responses.
ACKNOWLEDGMENT
We thank Dr David Kaplan for help with the P13 kinase assays,
Gitte Jorgensen for excellent technical assistance, and Laura Martinez for help with the manuscript.
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