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Associated with Insulin Secretion Vesicles1
Department of Internal Medicine (M.M., S.W., P.M., M.O., P.A.H., H.S., A.P.), BG Kliniken Bergmannsheil, University of Bochum, Medical School, D-44789 Bochum, Germany; Division of Clinical Biochemistry (J.L.), University of Geneva, Medical School, CH-1211 Geneva, Switzerland
Address all correspondence and requests for reprints to: Prof. Dr. Andreas Pfeiffer, Medizinische Klinik und Poliklinik, Berufsgenossenschafliche Kliniken Bergmannsheil, Universitätsklinik, Bürkle de la Camp-Platz 1, D-44789 Bochum, Germany. E-mail: Andreas.Pfeiffer{at}rz.ruhr-uni-bochum.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, 
,
) occur in insulin-secreting cells, we amplified all types of CaM
kinase II by RT-PCR and found the ß3-, -,
2- and 6-subtypes in RINm5F insulinoma
cells. None of the other 8 -subtypes was present. Antibodies
generated against the bacterially expressed association domain of the
2-subtype recognized the recombinant and
-subtypes. In INS-1 and RINm5F cells, as well as freshly isolated
rat islets, only a 55-kDa protein corresponding in size to the
2-subtype expressed in NIH3T3 fibroblasts was detected.
The 2-subtype therefore appears to represent the
predominant subtype of CaM kinase II present in insulin secreting
cells. The enzyme was primarily associated with cytoskeletal
structures, and very little was present in the soluble compartment or
detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of
its subcellular distribution was performed by sucrose and Nycodenz
density gradient fractionation of INS-1 cells and detection of CaM
kinase II by immune blots. The enzyme codistributed with insulin
used as a marker for secretory granules but not with the lighter
synaptic-like microvesicles detected with an antibody against
synaptophysin, plasma membranes (syntaxin 1), lysosomes
(arylsulfatase), or mitochondria (cytochrome c
oxidase).
CaM kinase II 2 thus is identified as the subtype
associated with insulin secretory granules and is likely to be involved
in insulin secretion.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The multifunctional CaM kinase II is present in most cell types and is
an important mediator of Ca2+-dependent signal transduction
(10, 11, 12). Four isoenzymes have been cloned and characterized named ,
ß, and (10, 13, 14, 15, 16). These isoforms are highly conserved and
have over 90% amino acid sequence homology in the N-terminal kinase
domain and in the regulatory domain comprising the
Ca2+/calmodulin binding sequence that is located in the mid
of the molecule (12).
The association domain comprises the C-terminal 2/5 of CaM kinase II and is less conserved among the different isoenzymes. In fact, for each isoform 2–10 splice variants have been described that appear to be important for the subcellular localization of the enzyme as indicated by the work of Srinivasan and colleagues (17, 18, 19, 20, 21, 22, 23, 24).
In the present report, we investigated which of the isoenzymes of CaM
kinase II occur in insulin secreting cells and define the splice
variants of CaM kinase II present. We moreover developed an
antibody against the bacterially expressed association domain of CaM
kinase II and characterized protein expression of the isoforms
as well as its subcellular distribution.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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35S]dATP and [32P]ATP were
from Du Pont de Nemours (Dreieich, Germany). INS-1 cells (25) were
kindly provided by C. Wollheim (Geneva, Switzerland). Human CaM kinase
B complementary DNA (cDNA) was a gift from H. Schulman
(Stanford, CA).
Enzyme preparation
Rats were anesthetized with diethyl ether and killed by cervical
dislocation. RINm5F and INS-1 cells where cultured in RPMI 1640 (Life
Technologies, Gaithersburg, MD) containing additions as described (25, 26). Tissues or cells were homogenized in ice-cold homogenization
buffer using a Polytron. The buffer consisted of 50 mM
Tris/HCl pH 7.4, 2 mM EDTA, 10 mM EGTA, 1
mM phenyl methyl sulfonyl fluoride (PMSF) added freshly, 1
mM dithiothreitol, 100 µM leupeptin, 0.3
µM aprotinin.
Assay of kinase activity
CaM kinase II assays were performed using autocamtide II
(KKALRRQETVDAL) as substrate (27) in 50 µl volume containing 10 µl
enzyme preparation (1–5 µg protein), 50 mM Tris, pH 7.5,
20 µM ATP, 10 mM MgCl2, 0.1
mM CaCl2 in excess of EGTA, 1 µM
calmodulin, 1 mg/ml BSA, 0.2–2 µCi [32P]ATP/assay
and 1–40 µM autocamtide II. Controls contained calcium
but no calmodulin. By determining background in the presence of
Ca2+, only calmodulin-dependent activity was assayed and
interference by Ca2+-activated calmodulin-independent
kinases was eliminated. The assay was started by addition of either
enzyme preparation or [32P]ATP. The reaction was
linear over 5 min at 21 C. After 3 min, the reaction was stopped with
25 µl 20% TCA, and an aliquot was applied to Whatman P81
phosphocellulose paper that was washed according to Roskowski (28) and
dried before determination of radioactivity by Cerenkov-counting in a
Beckmann LS 6000 counter.
RT-PCR
Total RNA was purified from RINm5F-cells by the method described
by Chomczynski and Sacchi (29), and about 1 µg was used for reverse
transcription using M-MLV reverse transcriptase (200 U, Life
Technologies) and random hexamer primers (Boehringer).
PCR was performed with Taq-DNA-polymerase (Cetus or
Boehringer) over 30 cycles either with two primer pairs specific for
the ß-subtypes (pair 2 and 6) or with a primer pair allowing
amplification of the -, - and -subtype (pair 1).
For cloning of the complete cDNA sequence of the subtype , three
overlapping PCR-amplificates (pairs 3–5) were restricted in the
overlapping region by Acy I or SstI and religated with
T4-DNA-ligase. The result was controlled by sequencing. To facilitate
cloning and to obtain more positive clones, the CloneAmp-Kit (Life
Technologies) was used for primer-pair 5. After PCR, the samples were
separated on a 1.5% agarose gel.
Primer sequences, annealing temperatures, and final
Mg2+-concentrations are shown in Table 1
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Sequencing
Sequencing was performed after subcloning by the
didesoxynucleotide-chain-termination-method described by Sanger (31)
using Sequenase 2.0 (USB) and [35S]dATP. Both strands
were sequenced.
Restriction analysis of the PCR products
To determine the subtypes present, restriction analysis of
amplificate 1 (Table 1
) was performed using different endonucleases
(Bst XI, EcoRV, DraII, HaeIII,
MaeII, and XhoII). This approach is based on the
differences of the sequences of the subtypes , and . The
expected restriction fragments are shown in Table 2.
Before restriction analysis, PCR product 1 was purified by agarose
gel-electrophoresis using a glass-matrix (Gene-Clean, Bio 101).
Restriction products were separated on polyacrylamide gels. Silver
staining was used for detection (see Fig. 2).
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) was designed for subtype
ß3 (32) that amplified a 215-bp sequence as predicted and
yielded the correct fragments after restriction with AluI
(116/84/15), FokI (120/95) and MboII
(108/107).
Bacterial expression and generation of polyclonal antibodies
To obtain an antigen for antibody production the C-terminal 194
amino acids of CaM kinase II subtype 2 [longer
amplificate of primer pair 5, nomenclature according to (19)] were
cloned into a bacterial expression vector pQE 31 (Quiagen, Hilden,
Germany) by restriction with BamHI and PspAI.
Using the N-terminally fused hexahistidine tag, the bacterially
expressed protein was purified by denaturing chromatography on a metal
chelate-affinity column loaded with Ni+ according to the
instructions of the producer. The purified protein was used as
immunogen for sc immunization of three rabbits in the presence of
complete Freund’s adjuvans. Four booster immunizations with incomplete
Freund’s adjuvans followed. The resulting antiserum recognized 500
fg/lane of the protein used for immunization on Western blots.
To assess the cross-reactivity of the antibodies to the -subtype,
the cDNA of human CaM kinase II B (20) that is 100%
homologous to the rat protein sequence was cloned into the
BamHI and SacI sites of pQE 30 (Quiagen) and
expressed in bacteria. The antibody recognized the
B-isoform in immune blots of bacterial lysates.
Immune blot
Cells or tissues were homogenized in homogenization buffer as
described above. Quantification of protein content was performed
according to Bradford using the modification of Stoscheck (33). The
homogenized protein was denatured for 5 min at 94 C in SDS-sample
buffer (0.0625 M Tris-HCL pH 6, 8, 2% SDS, 10% glycerol,
5% 2-mercaptoethanol, 0,002% bromphenolblue, 5 M urea)
just before SDS-gel-electrophoresis according to the method of Laemmli
(34). Molecular weight markers were from Sigma or from biotinylated
ECL-mol wt markers from Amersham. Semidry protein transfers onto
PVDF-membranes (Immobilon P, Millipore) were done as described by
Burnette (35). Transfer efficiency was checked by Ponceau-staining of
the membrane. After blocking in standard PBS containing 5% nonfat dry
milk (Gluecksklee GmbH, Munich, Germany) and 0,05% Tween-20 the
membrane was incubated in a 1:500 dilution of the immune serum in PBS
containing 0.5% nonfat dry milk. After two wash steps with standard
TBS protein A conjugated to alkaline-phosphatase (Calbiochem) was used
for detection. Visualization was done by BCIP/NBT staining
(bromchlorindoylphosphate/nitroblue-tetrazolium) according to a
standard protocol (36). Specificity of detection was demonstrated by
preadsorption of the antibody with the immunogen over 1 h at 4
C.
The detection of cellular marker enzymes by immune blots and the
detection CaM kinase II (see Figs. 3b and 5a) was performed with the
ECL system from Amersham using streptavidin-HRP for detection as
described (37).
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).
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Nycodenz (Nyegaard Diagnostica, Norway) step gradients (40) were performed using a detailed protocol kindly provided by B. A. Wolf as described (41). Briefly, 27.6% (wt/vol) Nycodenz stock solution was mixed with homogenization medium (0.3 M sucrose, 10 mM N-morpholino-ethanesulfonic acid, 1 mM EGTA, 1 mM MgSO4, pH 6.5) in the ratio 64/36 (A), 32/68 (B), and 16/84 (C). INS-1-cells were homogenized with a tight fitting glass Teflon potter in homogenization medium and layered over the Nycodenz media in a centrifugation tube. After spinning 100,000 x g for 60 min in a swing out rotor the interphase between A/B containing the enriched insulin granules and the interphases B/C and C/homogenate (containing enriched plasma membranes) were collected by centrifugation washed by resuspension and centrifugation in 0.25 M sucrose, 10 mM N-morpholino-ethanesulfonic acid, pH 6.5, and analyzed by Western blot as described above. Determination of arylsulfatase and cytochrome c oxidase (39) as lysosomal and mitochondrial markers showed a 2- to 4-fold reduction in the granule fraction relative to the initial homogenate. Insulin was enriched 8-fold in the granule relative to the homogenate fraction. Insulin was determined by RIA using antibodies against human insulin (Sigma) and [125I]human insulin with rat insulin as standard (Novo, Copenhagen, Denmark) as described (42).
Islet preparation
Islets were prepared by the collagenase method as described
(43). Collagenase (0.3 mg/ml, Serva, Heidelberg, Germany) in RPMI 1640
was injected into the ductus pancreaticus of 200 g anesthetized
Wistar rats. After digestion and repeated centrifugation islets were
hand picked under a stereo microscope. For each experiment 500–800
islets were homogenized and analyzed by immune blot as described
above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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through . Three pairs of PCR primers
were designed that were expected to amplify all CaM kinase II subtypes.
The primer pairs yielded amplificates of the expected length of 491 bp
for the ß1- and ß2-specific primer pair 2,
215 bp for the ß3-primer pair 6 and 219 bp for primer
pair 1 (Table 1). The ß1/ß2-specific primer
pair amplified the expected 491 bp fragment in rat brain mRNA but not
in insulinoma cells suggesting the absence of CaM kinase
ß1/ß2 (Fig. 1, lanes 1–4).
A primer pair designed to detect expression of the
ß3-subtype amplified the expected 215 bp sequence in cDNA
from RINm5F cells (Fig. 1, lanes 5–7). Digestion with AluI,
FokI, and MboII yielded the expected fragments,
confirming that the correct sequence was amplified (data not shown).
Primer pair 1 was expected to amplify CaM kinase II subtypes ,
and . Rat brain was used to demonstrate that these CaM kinase II
subtypes can be shown by this approach although there were mismatches
with the - and -subtypes (44). The subtypes expressed were
determined by analyzing the restriction pattern that differs between
the subtypes due to minor sequence variations (Table 2). The absence of
restriction of the amplification product of primer pair 1 by
DraII in insulinoma cells suggested the absence of the CaM
kinase II subtype . The fragment pattern of the amplificate of
primer pair 1 obtained with the restriction enzymes BstXI
and EcoRV and the complete cut of XhoII suggested
the presence of and subtypes in insulinoma cells (Fig. 2 and Table 2). The restriction patterns obtained with
the remaining enzymes confirmed this result.
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has been described
(18, 19, 23, 45). We now examined which of these splice variants occur
in RINm5F-insulinoma cells and which are absent. To this end, the
C-terminal region containing both variable domains was amplified by PCR
(primer pair 5) and subcloned for detailed analysis by sequencing.
Of the known subtypes of CaM kinase II , the mRNAs of only two
subtypes were detectable in RINm5F cells: both completely lacked the
upper variable domain (bp 985-1086). The C-terminal variable domain (bp
1536–1624) was either present or deleted. Taken together, these
results demonstrate the presence of the subtypes 2 and
6 according to the previously published nomenclature
(19).
The demonstration of CaM kinase II -mRNA does not necessarily prove
the expression of the protein. We therefore raised polyclonal
antibodies against the association domain of CaM kinase II
2 that was generated by expression of the C-terminal 194
amino acids in bacteria. The resulting antibody recognized a 55-kDa
protein in Western blots of RINm5F cell homogenate. The 55-kDa band was
completely eliminated by pretreatment of the antiserum with the
immunizing protein (Fig. 3a). The protein corresponds in
size exactly to CaM kinase II 2 expressed in NIH3T3
cells using an ecotropic retroviral expression system (detailed report
in preparation). Subtypes 6 and (21) expected to
migrate at 53 kDa or at approximately 59 kDa, respectively, were not
detected at the protein level. The antibody detected bacterially
expressed CaM kinase II B (see Materials and
Methods).
In conclusion, the major subtype of CaM kinase II present in RINm5F
cells appeared to be the subtype 2. It was therefore of
interest to determine whether the same isoform was present in the more
differentiated cell line INS-1 and in normal rat islets. Immune blots
comparing RINm5F with INS-1 cells and with normal freshly prepared
islets showed that an immunoreactive band of the same size was present
in all three cell types. Preadsorption of the antibody with the
immunizing protein strongly reduced staining of the 55-kDa band,
suggesting that CaM kinase II 2 was detected (Fig. 3b).
Remarkably, the expression level of CaM kinase II was markedly
higher in Islets and in INS-1 cells as compared with RINm5F-cells.
To characterize the subcellular distribution of CaM kinase II
2, INS-I cells were fractionated into cytosol and a
membrane-cytoskeletal fraction. The pellet was extracted with 0.5%
NP-40 detergent leaving a cytoskeletal fraction. The entire protein of
each fraction, obtained from a total of 100 µg of cell-protein, was
analyzed by immune blot to enable a quantitative comparison of the
amount of CaM kinase II in each subcellular compartment (Fig. 4). The majority of CaM kinase II 2 was
located in the cytoskeletal fraction, and only very little of the
protein was cytosolic or in the detergent soluble membrane compartment.
Similar results were also obtained by analyzing 20 µg of
protein/fraction.
The amount of CaM kinase II enzyme activity was measured in these fractions using the highly specific substrate autocamtide II (27, 46). The activity of CaM kinase II in the crude cytosolic fraction of RINm5F cells at saturating concentrations of autocamtide II was slightly below 10 pmol/µg·min and 27 pmol/µg·min in the cytoskeletal fraction. The solubilized membrane fraction did not contain reproducibly measurable CaM kinase II activity .
To investigate the subcellular distribution of CaM kinase II in
INS-1 cells, we employed linear 0.25–2.0 M sucrose
gradients (n = 4). This method is well established for INS-1 cells
and allows a good separation of insulin secretory granules from
synaptic-like microvesicles and other membranes (37). Synaptotagmin was
used as a marker for plasma membranes, synaptophysin as a marker for
synaptic-like microvesicles, and arylsulfatase was used as a lysosomal
marker (Fig. 5, a and b). The insulin content served as
a marker for dense-core granules. CaM kinase II comigrated closely
with the fractions highly enriched in insulin secretory granules (Fig. 5, a and b). The other fractions, in particular synaptic-like
microvesicles and plasma membranes, had a low content of CaM kinase II.
Unfortunately, the lysosomal marker arylsulfatase comigrated with
insulin granules (Fig. 5b).
To achieve a clear separation of lysosomes and mitochondria from
insulin granules, the Nycodenz step gradient technique was employed
(40, 41). This allowed an 8-fold enrichment of insulin relative to the
initial homogenate while arylsulfatase and cytochrome c
oxidase were reduced to less than half the content of the original
homogenate. CaM kinase II 2 was 6- to 8-fold enriched in
the insulin granule fraction and almost undetectable in the plasma
membrane fraction (Fig. 5c).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and 2 and 6-mRNA
in RINm5F insulinoma cells and largely excludes the presence of and
ß1/2-subtypes. The occurrence of these subtypes might
have been expected because a previous study indicated expression of
and types of CaM kinase II in peripheral tissues, whereas the -
and ß1/2-subtypes were found in neuronal structures (10).
The ß3-subtype was described in HIT T15 and
MIN6-insulinoma cells (32). We did not employ isolated rat islets for
these experiments because islets contain non-ß-cells, the mRNA of
which will easily be amplified by RT-PCR.
We have further characterized variants of CaM kinase II at the RNA
level in insulinoma cells. One variant type expressed, CaM kinase II
2, lacked 34 amino acids immediately C-terminal to the
calmodulin binding domain, whereas the other, CaM kinase II
6, lacked this upper variable domain and additionally
the C-terminal 21 amino acids that distinguishes the originally
described -type from all other isoforms of CaM kinase II to
(10, 18, 19).
The presence of particular splice variants may be of relevance
regarding the role of CaM kinase II in insulin secretion. In brain
synaptosomes for example, CaM kinase II and ß are present and CaM
kinase II regulates the phosphorylation of synapsin I (47).
Synapsin appears to anchor vesicles to cytoskeletal components,
particularly to actin, and probably prevents the final steps of vesicle
transfer from this pool to the pool immediately available for release
(48). CaM kinase II binds to synapsin I and releases vesicles from
the cytoskeletal pool by phosphorylation of synapsin I. Although CaM
kinase II ß is largely homologous to CaM kinase II outside the
variable domains, it neither binds to nor phosporylates synapsin I
(47). The presence of the CaM kinase II ß-specific variable domains,
therefore, may prevent association to structures of the cytoskeleton.
The work of Srinivasan and co-workers (17) underlines the importance of
the association domain for intracellular targeting. Moreover, CaM
kinase II 2 might function similiarly to brain CaM
kinase II in insulin secretory cells. In fact, Matsumoto and
co-workers (9) recently characterized a synapsin-1-like protein in
MIN-6 insulinoma cells that was a substrate of CaM kinase II and became
rapidly phosphorylated upon stimulation of the cells with glucose, KCl
or tolbutamide.
To investigate the expression and subcellular localization of CaM
kinase II in more detail, we raised antibodies against the
C-terminal association domain of CaM kinase II 2
comprising 196 amino acids. We used this approach upon failure to
obtain high affinity antibodies against the -specific C-terminal 15
amino acids after immunization of four rabbits. The antibodies against
the rat CaM kinase II -association domain were of high affinity and
recognized the eukaryotic expressed subtype 2, several
splice variants of the -isoform extracted from different tissues in
Western blots (Möhlig, M., P. A. Horn, and A. Pfeiffer,
manuscript in preparation) and the bacterially expressed
B subtype. RINm5F- and INS-1-insulinoma cells contained
a single compatible band at 55 kDa that corresponds to the predicted
size of the 2-subtype and exactly comigrates with CaM
kinase II 2 expressed in NIH 3T3 cells (data in
preparation). The smaller 6-subtype and subtype were
not detected, suggesting predominant expression of the
2-subtype. At present, it is not clear whether the
ß3-isoform (32) is expressed at the protein level, as we
do not know the cross-reactivity of our antibody with this isoform. The
ß3-isoform would be expected to migrate at approximately
60 kDa (32).
Our data agree excellently with those of Matsumoto and co-workers (9),
who observed a 55-kDa isoform of CaM kinase II in MIN-6 cells using a
nonspecific antibody generated against rat brain CaM kinase II. Quite
remarkably, these authors did not observe other isoforms of CaM kinase
II with this nonspecific antibody supporting the notion that the
2-isoform appears to be the predominant subtype
expressed in MIN-6 cells. Norling and co-workers (49) purified a 54 kDa
and 57 kDa CaM kinase II activity from RINm5F-cells. The 54-kDa CaM
kinase II activity agrees excellently with 2-subtype,
whereas the nature of the 57 kDa activity remains to be identified.
Insulinoma cell lines may show altered expression of proteins
characteristically expressed in islets. The observation that CaM kinase
II 2-like immunoreactivity identical to that in INS-1
and RINm5F-cells is present in islets suggests that this isoform is
strongly expressed in islets. Remarkably, the expression level of CaM
kinase II 2 was markedly higher in islets and
INS-1-cells compared with RINm5F-cells. RINm5F-cells have a greatly
reduced insulin content compared with INS-1 cells and islet ß-cells.
Therefore, the insulin content appears to parallel the CaM kinase II
2 content that might be expected in view of the
association of the enzyme with insulin granules.
Intracellular targeting may enable protein kinases with broad substrate specificities such as CaM kinase II to achieve selectivity of action in vivo. A study of the subcellular localization of the enzyme showed that most of the immunoreactivity was present in the detergent insoluble cytoskeletal compartment, whereas little immunoreactivity was present in the membrane and cytosolic fraction. This was observed in RINm5F and INS-1 cells and agrees with data from Norling and co-workers (49) on the distribution of the CaM kinase II activity. The cytoskeletal association differs from the distribution of CaM kinase II in other cell types where the majority of the enzyme is present in the cytosolic compartment (12). However, the distribution in INS-1 cells is comparable with that in brain synaptosomes that contain CaM kinase II predominantly associated with the cytoskeleton (50), where it is thought to direct synaptic vesicle traffic and function (48) as discussed above.
A more detailed analysis of the subcellular localization showed
comigration of CaM kinase II with insulin secretion granules in
INS-1 cells. This suggests that the enzyme may be either a component of
insulin containing dense core granules or of associated proteins. In
fact, this localization fits very well with the demonstration of a
synapsin-like protein in ß-cells that may be expected to be
associated with secretory granules similar to its function in synapses.
Moreover, the association with cytoskeletal elements may point to a
role of CaM kinase II in the transport or release of vesicles from such
structures. For example, actin filaments are involved in exocytosis in
endocrine cells as shown by a stimulation of this process by low
concentrations of reagents that depolymerize actin and by inhibition of
exocytosis by antibodies against the actin-organizing protein fodrin
(51) or an actin disrupting botulinum toxin (52).
The identification of the CaM kinase II 2 as the major
isoform in insulinoma cells and of its association with insulin
secretory vesicles will allow both: overexpression and suppression
studies that should allow a more precise investigation of the role of
CaM kinase II in insulin secretion.
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Acknowledgments |
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B-vector, B. A. Wolf for detailed instructions on
Nycodenz insulin granule purification, V. Kolb-Bachhofen for help with
rat islet purification and L. Heilmeyer for support and
discussions. |
Footnotes |
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2 Performed part of this work in fulfillment of the requirements of
their theses.
3 Recipient of a thesis support award by the University of
Bochum.
Received September 9, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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