Biochimica et Biophysica Acta 1452 (1999) 1^11
www.elsevier.com/locate/bba
Downregulation of N CaM kinase II in human tumor cells
Robert M. Tombes
a
a;
*, Ross B. Mikkelsen b , W. David Jarvis c , Steven Grant
c
Massey Cancer Center and Department of Biology, Medical College of Virginia Commonwealth University, Richmond, VA 23298, USA
b
Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA
c
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA
Received 30 April 1999; received in revised form 20 July 1999; accepted 3 August 1999
Abstract
Over two dozen alternative splice variants of CaMK-II, the type II Ca2‡ /CaM-dependent protein kinase, are encoded from
four genes (K, L, Q and N) in mammalian cells. Isozymes of K and L CaMK-II are well characterized in brain; however, an
understanding of the relative endogenous levels of CaMK-II isozymes in a wide variety of non-neuronal cells has not yet been
described. In this study, we have demonstrated that CaMK-II consists primarily of the 54 kDa N CaMK-II (N2 or NC ) isozyme
in rodent fibroblasts. L and Q CaMK-II isozymes are minor and K CaMK-II was not expressed. The primary N CaMK-II in
human fibroblasts and the MCF10A mammary epithelial cell line was the 52 kDa N4 CaMK-II, an isozyme identical to N2
except for a missing 21-amino-acid C-terminal tail. N CaMK-II levels were diminished in both human and rodent fibroblasts
after SV40 transformation and in the mammary adenocarcinoma MCF7 cell line when compared to MCF10A cells. In fact,
most tumor cells exhibited CaMK-II specific activities which were two- to tenfold lower than in untransformed fibroblasts.
We conducted complementary CaMK-II studies on the NGF-induced differentiation of rat PC-12 cells. Although no new
synthesis of CaMK-II occurs, neurite outgrowth in these cells is accompanied by a preferential activation of N CaMK-II.
Endogenous N CaMK-II has a perinuclear distribution in fibroblasts and extends along neurites in PC-12 cells. These findings
point to a role for N CaMK-II isozymes in cellular differentiation. ß 1999 Elsevier Science B.V. All rights reserved.
Keywords: CaM Kinase II; Downregulation ; Tumor; Human; Localization; PC-12; Neurite outgrowth; Isozyme
1. Introduction
CaMK-II, the type II, multifunctional Ca2‡ /CaMdependent protein kinase, is expressed in most mammalian tissues examined [1^3]. The unique ability of
CaMK-II to respond to both the amplitude and frequency of Ca2‡ £uctuations [4^9] makes this enzyme
highly desirable as a means for decoding Ca2‡ signals. CaMK-II was discovered in neuronal tissues
* Corresponding author. Fax: +1-804-828-8079;
E-mail: rtombes@hsc.vcu.edu
where K and L CaMK-II gene products have been
shown to be functionally important in neuronal signaling [10]. CaMK-II has been placed at various
points in a putative signaling hierarchy leading to
cellular di¡erentiation, including p42MAPK activation
[11,12], regulation of gene expression [13] or phosphorylation of terminal substrates such as microtubule associated proteins [14^16]. Studies using
CaMK-II mutants, inhibitory drugs and peptides
support a role for CaMK-II in neurite outgrowth
[17^19] and in adipogenesis [20] as well as in cell
cycle control [9,21^25].
The multifunctionality of CaMK-II is the result of
0167-4889 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 8 8 9 ( 9 9 ) 0 0 1 1 3 - 5
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structural diversity; alternative splicing in the central
hypervariable region and in N CaMK-II at the Cterminus gives rise to at least 27 di¡erent known
variants, varying in size from approximately 50 to
65 kDa, including two K, six L, nine Q and ten N
[26^35]. All CaMK-II isozymes are capable of forming oligomers of 8^12 subunits and exhibit conserved
catalytic properties, including CaM binding [1,29].
CaMK-II expression is controlled in part by tissuespeci¢c gene expression: K CaMK-II is restricted to
the central nervous system [33,36^38], while L, Q and
N CaMK-IIs occur in both neuronal and non-neuronal cell types from yeast to man, including a wide
variety of pancreatic, leukemic, breast and other tumor cells [29^31,33,39^47]. Nonetheless, it is becoming increasingly clear that the structural di¡erences in
the variable regions confer di¡erential subcellular
targeting to locations such as the nucleus and the
cytoskeleton in order to confer variable function
[45,48^50].
Therefore, in order to characterize the role of
CaMK-II in cell growth and di¡erentiation, it is important to de¢ne the relative levels of expression of
CaMK-II isozymes and their locations in proliferating cells and in those induced to di¡erentiate. Such
determinations of the endogenous CaMK-II isozyme
levels has been limited to only a few studies [32,51].
This study has comprehensively identi¢ed the level
of endogenous CaMK-II isozymes in a wide range of
both human and rodent cells using newly available
gene-speci¢c immunoprecipitating antibodies. We
have found that N CaMK-II isozymes are the major
CaMK-IIs in embryonic ¢broblast cell lines, have a
perinuclear localization and are downregulated by
transformation. Complementary studies indicate
that N CaMK-IIs are preferentially activated in cells
induced to di¡erentiate.
2. Materials and methods
2.1. Cell lines and culture
The following cell lines are listed with tumor type
of origin and American Type Culture Collection
(ATCC) number (Manassas, VA). WI38: human embryonic lung ¢broblasts (CCL75); MCF10A: human
mammary epithelial cells (CRL 10317); NIH/3T3:
mouse embryo ¢broblasts (CRL 1658); SV40 virus
transformed WI38 (CCL75.1); MCF-7: human
mammary adenocarcinoma (HTB 22); SV40 virus
transformed NIH/3T3 (CRL6476); CEM: human
lymphoblastic leukemia (CCL 119); U937: human
myelomonocytic leukemia (CRL 1593); HL-60: human promyelocytic leukemia (CCL 240); H146: human small cell lung carcinoma (HTB 173); MB-231:
human mammary adenocarcinoma (HTB 26);
DU4475: human mammary metastatic cutaneous
nodule carcinoma (HTB 123); SK-BR-3: human
mammary adenocarcinoma (HTB 30); MB-157: human mammary carcinoma (HTB 24); PC-12: rat
pheochromocytoma (CRL 1721); HT-29: human colon adenocarcinoma (HTB 38).
Cells were grown in culture to log phase and then
used to prepare cell lysates. Cells were cultured on
polystyrene dishes or in suspension in polystyrene
£asks in DMEM or RPMI-1640 (BioWhitaker, Walkersville, MD) with 10% fetal bovine serum (GIBCO-BRL, Bethesda, MD), supplemented with penicillin/streptomycin in a 5% CO2 humidi¢ed chamber
at 37³C. MCF10A cells were cultured in DMEM/F12 containing 100 ng/ml cholera toxin, 25 Wg/ml insulin, 0.5 Wg/ml hydrocortisone, 20 ng/ml EGF and
5% horse serum [52]. PC-12 cells were grown on 0.1%
poly-L-lysine-coated dishes and induced to di¡erentiate by the addition of 50 ng/ml (2U1039 M) nerve
growth factor (NGF) or 1 mM dibutyryl cAMP
(dbcAMP). MCF10A cells were obtained by permission from the Karmanos Cancer Institute at Wayne
State University. The human neuroblastoma LAN5
cell line was used as described [53] and provided by
Drs. Geo¡rey Krystal and Susan Hines.
2.2. Whole-cell lysate preparation
In order to examine enzymatic activities and immunoreactive polypeptides, cells were grown to log
phase, harvested (with trypsin^EDTA for adherent
cells) and then rinsed centrifugally (2000Ug for
5 min) with ice-cold PBS (phosphate-bu¡ered saline)
containing 2.5 mM EGTA. Cellular pellets were resuspended in 3 volumes of ice-cold homogenization
bu¡er, which consisted of 20 mM Hepes (pH 7.4),
2.6 mM EGTA, 20 mM MgCl2 , 80 mM L-glycerol
phosphate, 5 mM NaF, 0.1 WM okadaic acid
(GIBCO-BRL), 0.1 WM calyculin A (GIBCO-BRL),
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0.1 mM dithiothreitol, 0.01 mg/ml each chymostatin,
leupeptin, aprotinin, pepstatin and soybean trypsin
inhibitor (Sigma, St. Louis, MO). Samples were
then sonicated (two 5-s bursts on ice), centrifuged
at 12 000Ug for 15 min at 4³C and either assayed
immediately or frozen and stored at 370³C. Lysates
prepared by sonication solubilized over 90% of the
total CaMK-II activity as measured by solution assays and immunoblots (data not shown).
2.3. CaMK-II assays
CaMK-II assays were conducted using phosphocellulose paper assays and autocamtide-2, a peptide
which is modeled on the autophosphorylation site of
CaMK-II [54]. Assays were conducted in a total volume of 25 Wl containing ¢nal concentrations of
20 mM Na Hepes (pH 7.4), 15 mM Mg acetate,
10 mM NaF, 20 mM L-glycerol phosphate, 0.2 WM
okadaic acid, 0.5 WM protein kinase A inhibitor peptide, 0.2 mM dithiothreitol (DTT), 0.5 WCi [Q-32 P]and 20 WM total ATP, 35 WM autocamtide-2 (substrate) and either 1.04 mM EGTA (3Ca2‡ ) or 1 WM
calmodulin plus 1.04 mM EGTA/2.0 mM CaCl2
(+Ca2‡ ). After 5^10 min at 32³C, 20 Wl was pipetted
onto P81 phosphocellulose (Whatman, Clifton, NJ)
paper squares which were air-dried for 1 min and
then washed ¢ve times in 500 ml 1% phosphoric
acid. Dried paper squares were quanti¢ed by Cerenkhov counting in a Beckman model LS 1801 scintillation counter. Autocamtide-2 [KKALRRQETVDAL]
was purchased from Peninsula Laboratories (Belmont, CA), calmodulin was from Boehringer
Mannheim (Indianapolis, IN), [Q-32 P]ATP was from
NEN-DuPont (Wilmington, DE) and all other reagents were from Sigma unless speci¢ed. Protein concentrations were determined by BCA assay (Pierce,
Rockford, IL) in triplicate. CaMK-II assay conditions were threshold optimized for Ca2‡ , CaM and
ATP concentrations and optimized for linearity with
respect to cytosol protein concentration and time of
assay.
To begin the reaction, cytosol was added to a ¢nal
protein concentration of 0.1^0.2 mg/ml protein and
assayed for 5^10 min under the following three conditions: (a) EGTA without substrate; (b) EGTA
with autocamtide-2 substrate; and (c) Ca2‡ /CaM
with substrate. Ca2‡ -independent activity was deter-
3
mined from the di¡erence between b and a while
total activity was determined from the di¡erence between c and a. CaMK-II autonomy was then calculated as the percentage of total activity which was
Ca2‡ -independent. Background activity (a) was typically 6 1% of total activity,
2.4. CaMK-II antibodies
Puri¢ed rabbit anti-CaMK-II antibody reactive
with all mammalian CaMK-IIs (catalog no. 06-396)
was used at 1 Wg/ml (Upstate Biotechnology, Lake
Placid, NY). This antibody was raised against two
peptides common to CaMK-IIs: CTRFTDEYQLFEEL (residues 7^20 in rat N CaMK-II) and
EETRVWHRRDGKWQNVHFHC (residues 514^
532 in mouse L CaMK-II). These antigens were 90^
100% identical when compared to both rat and human sequences. This antibody was unable to recognize native CaMK-II as determined by its inability to
immunoprecipitate rodent or human CaMK-II activity or its inability to detect CaMK-II via indirect
immuno£uorescence, presumably because the conserved epitopes are buried within the structure of
these enzymes in their native conformation. Genespeci¢c antibodies and their peptide sequence
antigens included L CaMK-II (RRDGKWQNVHFHCSGAPVAP), Q CaMK-II (KWLNVHYHCSGAPAAPLQ) and N CaMK-II (CIPNGKENFSGGTSLWQNI). The L antibody recognized rat brain
L CaMK-II and the Q antibody recognized ectopically expressed Q CaMK-IIs. These antibodies were
able to recognize native and denatured CaMK-II as
determined by immunoblots, immunoprecipitations
and indirect immuno£uorescence.
2.5. Immunoblots
Sonicated whole-cell lysates were separated on 6%
or 10% polyacrylamide gels using a `Mini-Protean'
gel electrophoresis system (Bio-Rad, Richmond,
CA). Proteins were then transferred to 0.2 Wm pore
size nitrocellulose sheets (Schleicher and Schuel,
Keene, NH) for 1 h at 100 V and blocked with
Tris-bu¡ered saline (pH 7.4), 0.1% Tween-20
(TBST) containing 2.5% nonfat dry milk, 2.5% bovine serum albumin and 2% normal goat serum for
1 h. Primary antibodies were diluted to 1 Wg/ml in
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2% BSA/TBST/2% normal goat serum and incubated
between 2 and 12 h with the nitrocellulose blot. Secondary antibody incubations were for 2 h at 0.5 Wg/
ml coupled to alkaline phosphatase or to biotin
(KPL: Kierkegaard Perry Labs, Gaithersburg, MD)
in 2% BSA/TBST. Streptavidin^alkaline phosphatase
at 1 Wg/ml (KPL) was used if necessary. Blots were
washed ¢ve times with TBST and then developed
with 0.25 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.25 mg/ml nitro-blue tetrazolium (Sigma)
in 0.1 M Tris, 0.1 M NaCl, 5 mM MgCl2 , pH 9.4.
Control immunoblots were performed in an identical
fashion except that the primary antibody was omitted. Although some background bands were visible,
the bands identi¢ed as CaMK-II in the text and ¢gures were not detected on this control blot.
2.6. Immunoprecipitations
Twenty Wg of sonicated whole-cell lysate was incubated with 0.5 Wg goat anti-CaMK-II speci¢c for
L, Q or N CaMK-II (Santa Cruz Biotechnology, Santa
Cruz, CA) for 2 h on ice in IP bu¡er which consisted
of 20 mM Hepes (pH 7.4), 2.6 mM EGTA, 20 mM
MgCl2 , 80 mM L-glycerol phosphate, 5 mM NaF,
0.1 WM okadaic acid, 0.1 mM dithiothreitol,
0.01 mg/ml each chymostatin, leupeptin, aprotinin,
pepstatin and soybean trypsin inhibitor, and 0.5%
Nonidet P-40 (NP-40). After 2 h, 1 Wg of biotinylated
rabbit anti-goat IgG (KPL) was added for another
2 h on ice. Finally, streptavidin-Magnespheres
(Promega, Madison, WI) were used to purify immune complexes through three washes with IP bu¡er. Pellets were resuspended in 20 Wl IP bu¡er lacking
NP-40 and then assayed for kinase activity.
2.7. Immuno£uorescence
Indirect immunolocalization of CaMK-II was conducted as follows. Cells were grown on 12-mm diameter #1 thickness glass coverslips and then ¢xed by
immersion in 100% methanol at 320³C for 3 min.
Coverslips were then inverted onto 35 Wl drops of
bu¡er for 30 min at 30³C. Coverslips were blocked
with TBST containing 5% bovine serum albumin and
2% normal rabbit serum. The primary CaMK-II
gene-speci¢c goat antibodies were used at 1 Wg/ml
in 2.5% BSA/TBST/2% normal rabbit serum. Sec-
ondary biotinylated rabbit anti-goat IgG antibodies
were used at 1 Wg/ml (KPL) in 2% BSA/TBST/2%
normal rabbit serum. Finally, streptavidin^Texas
Red at 1 Wg/ml in 2% BSA/TBST (Molecular Probes,
Eugene, OR) was used. Coverslips were rinsed three
times with TBST between each step, inverted onto
glass slides using mounting medium (KPL) and
sealed with ¢ngernail polish.
2.8. cDNA preparation and transfection
Full-length Q CaMK-II cDNAs were expressed in
the eukaryotic expression vector pSRK [27]. The fulllength human QB CaMK-II cDNA was obtained
from Dr. Howard Schulman, Stanford University,
and a partial cDNA for human QC CaMK-II was
cloned as described [33]. Full-length human QC
CaMK-II cDNA was prepared by swapping out the
242 bp variable region restriction fragment of QB for
the 173 bp QC PvuII fragment which £anks the variable domain.
Transfection of cDNAs encoding full-length
CaMK-II isozymes into NIH/3T3 ¢broblasts used
lipofectamine PLUS (Life Technologies, Grand Island, NY) for 3 h followed by culture for an additional 24 h. Cell lysates expressing these isozymes
were prepared as described above and used as standards for immunoblots.
3. Results
3.1. CaMK-II activity is downregulated in
transformed cells
CaMK-II enzymatic activity was determined from
cells harvested in the logarithmic stage of growth.
Assays were optimized as described (see Section 2)
and conducted in the presence or absence of Ca2‡ .
CaMK-II activity from these cells could be recovered
by CaM a¤nity chromatography and autophosphorylated, as previously described [22]. Previous
studies con¢rmed the expression of mRNAs encoding CaMK-IIs from many of these cells [33].
Ca2‡ /CaM-dependent CaMK-II speci¢c enzymatic
activity varied by tenfold from 0.7 to 7.0 nmol/min
per mg among the cells studied here (Table 1). These
values are within the range reported by other labo-
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Table 1
CaMK-II enzymatic activity by cell type
Cell type
n
3Ca
+Ca
Non-transformed cell lines and transformed counterpart
NIH/3T3 ^ embryonic ¢broblasts, mouse
SV-3T3 ^ SV40 transformed NIH/3T3
WI38 ^ embryonic lung ¢broblasts
SV-WI38 ^ SV40 transformed WI38
MCF10A ^ mammary epithelial
MCF7 ^ mammary adenocarcinoma
7
2
3
3
4
5
0.18 þ 0.05
0.18 þ 0.02
0.19 þ 0.01
0.27 þ 0.02
0.19 þ 0.04
0.15 þ 0.04
4.54 þ 0.30
2.61 þ 0.04
6.56 þ 0.21
3.24 þ 0.17
5.36 þ 0.34
2.68 þ 0.26
Leukemic cell lines
CEM ^ lymphoblastic
U937 ^ myelomonocytic
HL60 ^ promyelocytic
3
3
3
0.12 þ 0.01
0.14 þ 0.03
0.18 þ 0.02
0.92 þ 0.08
1.21 þ 0.15
1.48 þ 0.10
Other tumor cell lines
H146 ^ small cell lung cancer
LAN5 ^ neuroblastoma
PC12 ^ pheochromocytoma, rat
HT29 ^ colon adenocarcinoma
2
3
4
2
0.14 þ 0.02
0.23 þ 0.02
0.23 þ 0.02
0.32 þ 0.02
1.72 þ 0.22
3.68 þ 0.35
5.36 þ 0.25
6.64 þ 0.23
Cell lysates from the indicated cell lines (human, unless speci¢ed) were assayed in triplicate for Ca2‡ /CaM-dependent (+Ca) and -independent (3Ca) activity. Activity values are presented as nmol phosphate transferred to the peptide substrate, autocamtide-2, per min
per mg protein. Percent autonomy is calculated from the ratio of the two values.
ratories and re£ect the total level of CaMK-II [55].
Among these samples, rodent and human embryonic
or epithelial cell lines (WI38, MCF10A and 3T3) expressed high levels of CaMK-II. The HT-29 human
colon carcinoma and the PC-12 rat pheochromocytoma cell lines also expressed high levels. Cells which
expressed intermediate levels of CaMK-II included
tumor cell lines of breast (MCF7) and neuronal
(LAN5) origin. Cells which expressed low levels of
CaMK-II were the leukemic cells (CEM, HL60 and
U937). Repeatedly, we observed that SV40-transformation of both WI38 ¢broblasts (SV-WI38) and
NIH/3T3 ¢broblasts (SV-3T3) resulted in a twofold
decrease in the total CaMK-II activity. Similarly,
MCF7 mammary adenocarcinoma cells exhibited approximately 50% of the level of activity in MCF10A
mammary epithelial cells. Di¡erences between these
paired samples were highly signi¢cant (P 6 0.001).
The level of Ca2‡ /CaM-independent CaMK-II activity can be used as an indicator of in situ CaMK-II
activation. In contrast to total activity, the Ca2‡ -independent CaMK-II speci¢c activity varied only between 0.1 and 0.3 nmol/min per mg. When expressed
as a percentage of total activity (% autonomy), this
converted to values between 3% and 12%. Leukemic
cells consistently exhibited levels of CaMK-II
autonomy above 12%, possibly re£ecting the altered
Ca2‡ metabolism which occurs in some experimentally transformed cells or tumor derived cells [41,56^
58].
3.2. CaMK-II protein levels are decreased in
transformed cells
Immunoblots also revealed variations in total
CaMK-II activity among cell lines and tentatively
identi¢ed individual CaMK-II isozymes (Fig. 1). A
pan-speci¢c anti-CaMK-II antibody was used which
was reactive with conserved domains in the aminoand carboxyl-termini (NT/CT) of human and rodent
CaMK-IIs from all four genes (see Section 2). To
demonstrate its pan-speci¢city, the ¢rst two lanes in
Fig. 1A contain samples with known CaMK-IIs. The
two principal bands in rat cerebral cortex (lane A1)
are K CaMK-II at 52 kDa and L CaMK-II at 60
kDa; the 58 kDa minor band may represent LP
[59,60]. Cloned and expressed human QB and QC
also reacted well (lane A2). As described previously
[27], QB CaMK-II migrates with a Mr of 60 kDa in
spite of its predicted size of 58 372; QC CaMK-II also
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Fig. 1. CaMK-II isozymes by cell type. Whole-cell lysates (10 Wg unless indicated) from the following tissues and cell lines were prepared as described in Section 2, resolved by sodium dodecyl sulfate^polyacrylamide gel electrophoresis, transferred to nitrocellulose
and probed with the pan-speci¢c antibody reactive with conserved CaMK-II domains (A,B) and with gene product-speci¢c antibodies
(C^E). CaMK-II standards were from transient expression experiments in NIH/3T3 cells. Panel A: lane 1, 2.5 Wg rat cerebral cortex
(Br); lane 2, CaMK-II QB and Qc in 3T3 cells (2 Wg); lane 3, LAN5 (L5); lane 4, HT29 (HT); lane 5, CEM (C); lane 6, PC-12 (PC).
Panel B: lane 1, NIH/3T3 (3T3); lane 2, SV-40-NIH/3T3 (3/SV); lane 3, WI38 (WI); lane 4, SV40-WI38 (W/SV); lane 5, MCF10A
(M10); lane 6, MCF7 (M7). Panels C^E: lane 1, 2.5 Wg rat cerebral cortex (Br); lane 2, CaMK-II QB and Qc in 3T3 cells (2 Wg); lane
3, NIH/3T3 (3T3); lane 4, SV-40-NIH/3T3 (3/SV); lane 5, WI38(WI) ; lane 6, SV40-WI38 (W/SV) and lane 7, PC-12 (PC).
has an apparent mass (57 kDa) larger than its predicted size of 55 997.
In cell lines (Fig. 1, lanes A2^6 and B1^6), protein
immunoreactivity was proportional to total CaMKII activity, i.e., cells with high levels of CaMK-II
activity (HT29, PC-12, NIH/3T3 and WI38) exhibited the most immunoreactive bands, primarily at 52
or 54 kDa. These bands most likely represent NC (not
K) CaMK-II, whose observed Mr is in this range [29]
and whose mRNA was detected in these cells [33].
The major band was 52 kDa in the human cells
HT29 (lane A4), MCF10A (lane B5) and WI38
(lane B3) and the 54 kDa band in rodent NIH/3T3
(lane B1) and PC-12 cells (lane A6). The human tumor cells LAN5 (lane A3), H146 (not shown) and
MCF7 (lane B6) all displayed an almost identical
pattern of moderately intense 60, 54 and 52 kDa
bands. As with the activity assays, leukemic cells
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7
(CEM shown in A5) exhibited the lowest levels of
CaMK-II.
When paired transformed and non-transformed
samples were evaluated, the 52 kDa band in
MCF10A and WI38 cells was diminished in MCF7
and SV-WI38 cells, while the 54 kDa band in NIH/
3T3 cells was lost in SV-3T3 cells. CaMK-II bands in
the 60 kDa range were constant relative to the bands
in the 52^57 kDa range. These ¢ndings suggest that
decreased CaMK-II activity in transformed cells is a
result of decreased levels of speci¢c CaMK-IIs, particularly isozymes encoded from the N CaMK-II
gene.
3.3. N CaMK-II expression is decreased by
transformation
To evaluate whether the decrease in CaMK-II in
transformed cells was due to speci¢c CaMK-II gene
products, we used L, Q and N CaMK-II anti-C-terminal peptide antibodies (see Section 2). The L and the
Q CaMK-II antibodies reacted weakly with the
52 kDa K and the 60 kDa L from brain, but strongly
with the overexpressed human Q CaMK-IIs (Fig.
1C,D). This cross-reactivity was not surprising since
similar peptide epitopes were used to generate these
two antibodies (see Section 2). However, neither L
nor Q antibodies reacted with proteins in the cell lines
shown (Fig. 1C,D). The N CaMK-II antibody did not
react at all with the Q standards, but reacted well with
the 54 kDa putative NC CaMK-II in NIH/3T3, WI38
and PC12 cells (Fig. 1E). This antibody did not react
with the 52 kDa CaMK-II. This is consistent with
the existence of two NC CaMK-II variants, the
54 kDa protein with the C-terminal 21 amino acid
tail (N2 ) and the 52 kDa protein (N4 ) without this tail
[26]. These ¢ndings also indicate that N4 and N2 can
be co-expressed, but at di¡erent ratios in various cell
Fig. 2. CaMK-II autonomy in NGF-di¡erentiated PC12 Cells.
CaMK-II was immunoprecipitated with L and N gene-speci¢c
antibodies from cleared, sonicated PC-12 cells 48 h after NGF
treatment as indicated. Immunoprecipitated activity was then
analyzed for both Ca2‡ /CaM-dependent and -independent activity in triplicate. CaMK-II autonomy was calculated as a percentage of total activity.
types. The 57 kDa band in WI38 and PC-12 cells
likely represents other N CaMK-IIs with the C-terminal tail, as described [26]. As seen with the NT/CT
antibody (Fig. 1B), the 54 kDa N CaMK-II was decreased in transformed NIH/3T3 and WI38 cells relative to their non-transformed counterparts (Fig.
1E).
A decrease in N CaMK-II in transformed cells was
also demonstrated using immunoprecipitation assays.
The L and N CaMK-II anti-peptide antibodies e¤ciently immunoprecipitated active CaMK-II (Table
2), but only in rodent cells, since human N CaMKII was predominantly C-terminal tail-less. The
Q CaMK-II antibodies did not precipitate any signi¢cant activity from any cell type, although they very
e¤ciently recovered overexpressed Q CaMK-IIs (data
not shown). Consistent with immunoblots, SV40
Table 2
L, Q and N CaMK-II isozyme distribution among cell types
Gene
NIH/3T3
SV40-3T3
WI38
SV40-WI38
PC-12
L
Q
N
0.72 þ 0.04
0.06 þ 0.01
4.14 þ 0.21
0.58 þ 0.04
0.05 þ 0.01
1.63 þ 0.09
0.24 þ 0.01
0.03 þ 0.01
1.00 þ 0.04
0.12 þ 0.01
0.01 þ 0.01
0.38 þ 0.02
0.49 þ 0.04
0.00 þ 0.00
2.41 þ 0.19
CaMK-II was immunoprecipitated with gene-speci¢c antibodies from 10 Wg cleared, sonicated NIH/3T3, SV40-transformed 3T3,
WI38, SV40-transformed WI38 and PC-12 cells and then analyzed for Ca2‡ /CaM-dependent activity. Activity was then calculated as
nmol/min per mg for the original 10 Wg protein and assumed 100% recovery.
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Fig. 3. CaMK-II immuno£uorescence localization. CaMK-II was immunolocalized in logarithmically growing NIH/3T3 cells and in
PC-12 cells 72 h after NGF treatment with L, Q and N gene-speci¢c antibodies. Scale bar = 20 Wm. Images were exposed and printed
under identical conditions to show relative immunoreactivity.
transformation eliminated 60% of the N CaMK-II
activity found in NIH/3T3 cells but only 20% of
the L CaMK-II (Table 2). Similarly, SV40 transformed WI38 cells lost 63% of the N CaMK-II and
50% of the L CaMK-II.
3.4. N CaMK-II is activated in di¡erentiated
PC-12 cells
As a complement to the study of CaMK-II in
transformed cells, we evaluated changes in CaMKII in cells induced to undergo di¡erentiation. Since
several species of CaMK-II are found in the PC-12
cell line (see Fig. 1) and CaMK-II is reported to be
activated in these cells upon di¡erentiation [61], we
treated these cells with either nerve growth factor
(NGF) or dibutyryl cAMP (dbcAMP). Neurites
were observed extending from s 30% of cells 3 days
after treatment. Ample L and N, not Q CaMK-II was
immunoprecipitated from these cells (Table 2), but
the total activity and isozyme spectrum did not
change upon di¡erentiation (data not shown). In response to both stimuli, however, we observed an increase in the CaMK-II autonomy in total cell lysates
from 2% to 5% (Fig. 2). This was primarily the result
of the activation from 2% to 7% autonomy of
CaMK-II precipitated by the N CaMK-II antibody.
L CaMK-II activity increased to a lesser extent from
1% to 2% autonomy. Both increases were statistically
signi¢cant (P 6 0.005).
3.5. Localization of CaMK-II
The gene-speci¢c antibodies were also used to localize endogenous CaMK-IIs in NIH/3T3 and PC-12
cells. As expected, the N CaMK-II antibody exhibited
a much stronger reaction than L or Q antibodies.
N CaMK-II was found throughout both cell types,
but was concentrated in a perinuclear pattern in
NIH/3T3 cells and in PC-12 cells induced to di¡er-
BBAMCR 14538 1-10-99
R.M. Tombes et al. / Biochimica et Biophysica Acta 1452 (1999) 1^11
entiate with NGF (Fig. 3). L CaMK-II was found in
a similar but less intense perinuclear pattern whereas
Q CaMK-II was at background levels. Staining patterns observed in the absence of primary antibody
are not shown since they were indistinguishable
from those using the Q CaMK-II antibody. In PC12 cells, N CaMK-II was found along all neurite
branches. CaMK-II was excluded from the nucleus
in these cells, in contrast to previous reports on rat
and human ¢broblasts and glioma cells [62]. We do
not show undi¡erentiated PC-12 cells, since they are
much smaller than ¢broblasts and adhere poorly to
coverslips, particularly during the processing steps
for indirect immuno£uorescence.
4. Discussion
Although CaMK-IIs encoded by the K and L genes
are enriched and well characterized in tissue from the
central nervous system, the results presented here
have implicated N CaMK-II isozymes in the growth
and di¡erentiation of cells derived from peripheral
tissues. We have found that N CaMK-IIs are major
while L and Q CaMK-IIs are minor CaMK-II gene
products in ¢broblasts and in untransformed cell
lines derived from peripheral tissues. We have also
shown that oncogenic transformation results in the
preferential decrease in N CaMK-II protein levels and
thus activity. Finally, we have demonstrated that
N CaMK-II isozymes are activated in di¡erentiated
PC-12 cells and are found outside of the nucleus
along neurites.
These results are complementary to each other in
that N CaMK-II activity is associated with di¡erentiation in PC-12 cells and is decreased in a wide
variety of tumor cell lines, which in general are less
well di¡erentiated than non-malignant cells. The
known loss of cell adhesion and cytoskeletal anchoring as a general phenotype of tumor cells may be
consistent with a role for N CaMK-II in a common
pathway regulating cytoskeletal function. CaMK-II
is known to phosphorylate cytoskeletal proteins including the microtubule associated proteins, MAP2
and tau [63,64]. The substrates phosphorylated by
N CaMK-II isozymes in situ are not known.
Decreased CaMK-II levels in tumor cells is also
consistent with decreased G1 checkpoint control in
9
tumor cells [65], since CaMK-II has been implicated
in G1 cell cycle control [22,23,66]. In fact, it is known
that Ca2‡ /CaM-dependent pRb phosphorylation,
Ca2‡ /CaM-dependent DNA synthesis and Ca2‡ -dependent cell cycle progression are all lost in SV40transformed WI38 cells [57,67,68]. Certain CaMK-II
isozymes might therefore normally be involved in
transmitting G1-related Ca2‡ signals to activate cyclin-dependent kinases to phosphorylate pRB [66];
their loss during the course of transformation might
lead to a loss in the Ca2‡ -dependence of DNA synthesis. DAP kinase, a CaM kinase believed to be
involved in apoptosis and associated with the cytoskeleton, is also downregulated in a variety of tumor
cells [69].
Other laboratories have also reported the prevalence of N CaMK-IIs in mammalian tissues. For example, the 54 kDa NC (N2 ) CaMK-II is the predominant N isozyme in rat insulinoma cells [70] and
N CaMK-II mRNA is the predominant CaMK-II
gene product in PC-12 cells [17]. 54 and 52kDa
N CaMK-II isozymes have also been reported to be
the porcine cardiac sarcoplasmic reticulum-associated CaM-dependent phospholamban kinase [51].
The nuclear targeted NB CaMK-II is a major isozyme
in cardiac tissue and is believed to directly in£uence
gene expression [13,28,29]. Previous reverse transcription^polymerase chain reaction ¢ndings from
this laboratory indicated that no other N CaMK-II
central domain variant was produced in any of the
cells in this study except NE from LAN5 neuroblastoma cells [33].
Our ¢ndings predict that overexpressed N CaMKII isozymes might associate with the cytoskeleton
and promote di¡erentiation. Experiments to determine the direct in£uence of various ectopically expressed N CaMK-II isozymes on di¡erentiation and
neurite outgrowth are under way. In the H-7 neuroblastoma cell line, it has been reported that
L CaMK-II is more capable of inducing neurite outgrowth than K CaMK-II [19]. Such experiments need
to be conducted with other CaMK-II isozymes
known to be expressed in these cells.
The cells in this study which expressed the highest
levels of CaMK-II were distinguished by the predominant expression of non-nuclear N CaMK-II isozymes. Both 52 NC (N4 ) and 54 kDa NC (N2 ) isozymes
exist in these cells, but their ratio varies. In human
BBAMCR 14538 1-10-99
10
R.M. Tombes et al. / Biochimica et Biophysica Acta 1452 (1999) 1^11
cells (HT29, WI38, MCF10A), the ratio favors the
52 kDa NC (N4 ) CaMK-II whereas in rodent cells, the
54 kDa NC (N2 ) isozyme is the principal isozyme
(NIH/3T3, PC-12). The regulation or function of
the 21-amino-acid C-terminal tail is not yet known.
Cells which expressed intermediate levels of
CaMK-II included solid tumor cell types of breast
and neuronal origin, which had previously been
shown to express Le or LPe CaMK-II [33] and which
have observed Mr of 58 and 56 kDa [60]. Neither L3
CaMK-II, a 65 kDa (predicted) isozyme cloned from
pancreatic, testicular and islets of Langerhans cells
with an SH3-domain binding site [31,46], nor Lm
CaMK-II, a 72 kDa sarcoplasmic reticulum isozyme
[34], was found in any of the cell types examined in
this study. Full length L CaMK-II has been shown to
associate with actin and micro¢laments [49,50], but
the L CaMK-II antibody described here did not reveal such a pattern.
It was notable that Q CaMK-IIs comprised so little
of the activity and total protein within cells in spite
of the prevalence and diversity of mRNAs encoding
these CaMK-IIs in most cells [27,30,32,33]. The conclusion that Q isozymes are minor relative to
N CaMK-IIs has also been reached in smooth muscle
cells where Q CaMK-IIs have been studied extensively
[28,32,71]. This does not diminish the importance of
these or any other minor isozyme since CaMK-IIs
speci¢cally targeted within the cell could be locally
concentrated to function in very speci¢c roles.
In summary, the evidence presented points to an
active role for non-nuclear N CaMK-II isozymes in
the process of di¡erentiation. Future studies attempting to characterize the functional consequences of
overexpressed CaMK-II isozymes on cellular di¡erentiation and the identi¢cation of their substrates
and downstream signaling targets are now possible.
Acknowledgements
The authors would like to thank Dr. Howard
Schulman, Stanford University, for sharing CaMKII cDNA constructs and to Lora B. Kramer for tissue culture assistance. Partially puri¢ed rat brain
CaMK-II was kindly provided by Dr. S.B. Churn,
Virginia Commonwealth University. This work was
supported by a grant from the A.D. Williams Com-
mittee at Virginia Commonwealth University, Grant
J-448 from the Kate and Thomas Je¡ress Foundation Trust (R.M.T.) and Grant R01 CA63753 (S.G.,
W.D.J.).
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