Vol.
167,
March
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
RESEARCH
Pages
1990
1316-1325
INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF ASSEMBLYDISASSEMBLY OF NEUROFILAMENTS IN VITRO Yasunori
Gonda+, + Kitamura+,
Shinobu
Kimiko
Nishizawa,
Yasushi
Minouran,
and Laboratory
of Experimental
Aichi
§Life
Masaki
Cancer
Center
Information
Inagaki
Radiology Research
Shoji
Ando',
Yoshimi
Nishi§,
* and qBiophysics
Institute,
Nagoya
464, Japan
Analysis
Center,
Unit,
Chikusa-ku,
Komaki 485,
Japan
Received February 15, 1990 Protein kinase C phosphorylated the major mammalian neurofilament protein (NF-L) with ~3 mol phosphate per mol protein. The phosphorylated NF-L no longer formed the filaments. Sequential analysis of the tryptic phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-27, Ser-33 and Ser-51 were phosphorylated by protein kinase C. These findings contribute toward elucidation of mechanisms 01990 Academic Press, Inc. regulating the functions of neurofilaments.
Neurofilaments
are
neuronal
cytoskeleton
neuronal
size
major
and serve
and shape.
electrophoresis
showed that
polypeptides
with
160,000
(NF-M),
and 68,000
as
filaments
Life Science Research Co., LTD., Samejima,
*To whom correspondence
and Biological should
0 1990 by Academic Press, Inc. in any form reserved.
of reproducrion
1316
200,000
and the
of
gel consist (NF-H),
In reconstitution two high-
Laboratories, Asahi Fuji 416, Japan. Laboratories,
be addressed.
0006-291X/90$1.50
the
determinants
sulfate
masses of
(NF-L)(l-5).
only
$Present address: Medical Nagano 396, Japan.
Copyright AI1 rights
important
of
mammalian neurofilaments
molecular
NF-L formed
+Present address: Chemical Industry
components
Sodium dodecyl
three
experiments,
structural
Ina,
of
Vol.
167,
No.
3, 1990
molecular
weight
self-assembly,
proteins although
filaments core
BIOCHEMICAL
formed
of
the
Sihag
et
can
serve
vivo
(6).
al. as
both
by NF-L
that
a substrate
for
is
significance the
were
RESEARCH
showed
readily Thus,
action shows
of of
that
the
COMMUNICATIONS
evidence
incorporated NF-L
of into
may be the
filament-forming
protein
a paucity
the
central
kinase
in
protein
the
C, in
data
kinase
protein, vitro
related
kinase
C on NF-L.
protein
kinase
C stoichiometrically
NF-L
and that
kinase
phosphorylation
functional of
Experimental
the
NF-L and
to
in
the
C phosphorylation,
protein
phosphorylates follows
and NF-M)
(4,5).
reported
As there
we obtained
(NF-H
BIOPHYSICAL
filaments.
functional examined
AND
alteration specific
we
The evidence
by this domain.
Procedures
Purification of proteinsNF-L was purified from porcine brain white matter by the method of Liem and Hutchison (5) with some modifications (7). Protein kinase C from the rat brain was prepared by the method of Inagaki et al. (8). Phosphorylation of NF-LNF-L (1 mg/ml) was dialyzed against 5 mM Tris-HCl, pH 8.8, 2mM EGTA, 1 mM PMSF and 50 mM 2mercaptoethanol (Buffer A) for 24hr at 4OC for phosphorylation of the soluble form of NF-L, or against 10 mM PIPES, pH 7.0, 0.15 M NaCl, 2 mM EGTA, 1 mM PMSF and 50 mM 2-mercaptoethanol (Buffer B) for 6 hr at 37OC for phosphorylation of the filamentous form of NF-L. Both forms of NF-L (0.15 mg/ml) were ted by incubation with 5 ug/ml of protein kinase C, ~h~s~~o~?32p-~Tp 0.3 mM MgC12, 25 mM NaCl and 10 mM PIPES (pH 7:01
(9-11).
’
Electron microscopyIntermediate filament preparations were placed directly on carbon film-coated specimen grids and stained with 1 % or 2 % uranyl acetate or 2 % sodium phosphotungstate (pH 7.2). Phosphopeptides from35 adioactive NF-LSoluble NF-L (2 mg) was incubated with [vP] ATP and protein kinase C (40 pg) in an 8-ml mixture, as described above. The phosphorylated NF-L was precipitated with 10 % trichloroacetic acid and collected by centrifugation for 30 min at 10,000 x g at 4OC. The precipitated NF-L was dissolved in 7 M urea (3 ml), and dialysed overnight against 10 mM Tris-HCl (pH 8.6) at 4 OC. To the sample solution, we added 400 1-11 of 250 mM Tris-HCl (pH 7.5) and the final protein concentration was adjusted to 0.5 The phosphorylated NF-L mg/ml with 25 mM Tris-HCl (pH 7.5). (0.5 mg/ml) in 25 mM Tris-HCl (pH 7.5) was treated with L-ltosylamide-2-phenylethyl chloromethyl ketone-treated trypsin A 2 ml sample of l/25 (w/w) of NF-L) at 37OC for 2 h. (Sigma, the reaction mixture was applied to a Zorbax C8 (0.46 x 20 cm) column attached to a Waters HPLC system (model 510 pumps, a model 490 detector and an automated gradient controller) and 1317
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was
eluted with a linear gradient of IO-50 % acetonitrile in % trifluoroacetic acid over 70 min followed by a further linear gradient of 50-80 % acetonitrile in 0.1 % trifluoroacetic acid over 20 min at a flow rate of 0.8 ml/min. Elution of the fragments was monitored by UV at 230 nm and by the radioactivity of each fraction (0.8 ml). Purification of fragmentsThe radioactive fractions were applied to an anion exchange column of TSK gel QAE-2SW (0.46 x 25 cm) and were eluted with a linear gradient of O-O.5 M NaCl in 20 mM Tris-HCl (pH 7.5) over 40 min at a flow rate of 0.8 mllmin. Each radioactive fraction obtained was then applied to a Zorbax C8 (0.46 x 20 cm) column and was eluted with the same linear gradient used in the first fractionation of the at the same flow rate. Radioactive fractions phosphopeptides, obtained were lyophilized and stored at 4OC. Phosphoamino acid analysisProtein kinase C phosphorylated NF-L was subjected to acid hydrolysis in 6 N HCl for 1.5 hr at llo"c. The phosphoamino acids were resolved by electrophoresis at pH 3.5 on a cellulose thin layer plate, as described (9). Amino acid analysisPurified phosphopeptides (0.5-I nmol) were subjected to 6 N HCl hydrolysis in vacua at 110 OC for 24 hr. Amino acid analysis was performed by reverse-phase HPLC of phenylthiocarbamoyl derivatives (12) using the Waters Pica-Tag system. Sequence analysisA I%7 n mol sample of the purified fragments dissolved in 0.1 % trifluoroacetic acid was analyzed using an ABI 470A gas-phase sequencer equipped with an ABI 120A on-line PTH amino acid analyzer, using the 03R PTH program. 0.1
Results Protein of
kinase
NF-L.C of
shown
in
protein the
IA.
incorporated Protein
protein
kinase
a more
of
NF-L
NF-L
over
4 hr.
was
mol
of Thus,
the
for
protein
phosphorylated
competence
of
the
was
The amount
of
polymerized
with
increases
in
the
phosphate
protein
in 1318
the
as
used
phosphate
filamentous
kinase
NF-L
kinase NF-L (Fig.
1 mol
of
the
was
end of
soluble
is substrate
was
4 hr. form
form
of
of
NF-L
NF-
was
C.
by protein
polymerization analyzed.
as
into
the the
NF-L
phosphate
of
molecule'at
substrate
NF-L
of
of
used
filament
C, 21
by protein forms
was
~3 mol
NF-L
assembly-disassembly
phosphorylation
C phosphorylation for
the
filamentous
kinase
mol
appropriate soluble
and
When the
per
L continued
of
C incorporated
for
and
When soluble
molecule.
substrate
course
soluble
kinase
NF-L
The
time
both
Fig.
Discussion
C phosphorylation
The
kinase
and
kinase C-treated
C and NF-L
(ppt)
decreased
IB).
The
same
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B, soluble NF-L SP
SP
5
Tii
Fig
set
of
of
NF-L.
1.
finding
The
of
90
5
60
15 30 The(min)
is
in
good
agreement
of
in
2. C acts
the
of
These as
dissolved
for NF-L
120
90
with
NF-L
the
results
the
filament
a regulator
from
slow by
phosphorylated are
the
240
120
140
NF-L
rate
NF-L
form
filament
(Fig.
protein
interpreted of
filamentous
manner
in a time-dependent
slowly,
findings
in
30 60 Time(min)
NF-L
was performed
amount
Typical
kinase
SPSPSP
C, filamentous
(nlln)
experiments
phosphorylation
Fig.
15
SP
Time course of phosphorylation of NF-L by protein kinase C (A) and effect of phosphorylation on polymerization competence of soluble NF-L (B) and on the state of filamentous NF-L (C). A; phosphorylation of soluble and filamentous NF-L is represented by open circles and closed circles, 30, 60, 90, 120 and respectively. B; after 0, 5, 15, 240 min incubation with protein kinase C, this assay mixture was passed through a Millipore filter (0.22 to remove vesicles of phosphatidylserine and was pm) then dialyzed against Buffer B containing 7 M urea polymerization for 12 hr at 4OC and then against Buffer B for 6 hr at 37OC to promote filament formation. After centrifugation at 100,000 x g for 30 min, supernatants (s) and precipitates (p) were subjected to 10 % SDS-PAGE. C; after 0, 5, 15, 30, 60, 90, 120 and 240 min incubation with protein kinase C, the reaction mixtures were directly centrifuged at 100,OOOxg and then supernatants (s) and precipitates (p) were subjected to 10 % SDS-PAGE.
the
increased
SPSP
c).
1
of kinase
samples to
This
mean
C. are
shown
that
assembly-disassembly
protein of
vitro. Phosphorylation
identify
the
sites
site
located
on
phosphorylated
the
head
by protein 1319
domain-
kinase
To
C,
NF-L,
Vol.
167,
No.
radioactive
As
NF-L
subjected shown
in
Fig.
digested
3,
the
acid
COMMUNICATIONS
The in of at
Table
porcine NF-L
for
1.
NF-L
residues
acid In
purified
by anion
a comparison
(13),
peptides
30-36,
23-29,
exchange
under
radioactive
gas-phase
sequences
was
several
as described
1320
material column.
separated purified
as described
amino
and the
a reverse-phase
further
Each
analyzed
compositions,
listed
with
HPLC columns,
was
Procedures".
located
was
Procedures".
phosphopeptide
sequence
RESEARCH
trypsin
HPLC procedure
each
re-reverse-phase
amino
BIOPHYSICAL
with
HPLC equipped
and
"Experimental
are
was to
phosphopeptides and
AND
2. Electron microscopy of the negatively stained NF-L samples. A,B; polymerization competence of nonphosphorylated (A) and protein kinase C C,D; the state of phosphorylated (B) soluble NF-L. non-phosphorylated (C) and protein kinase C phosphorylated (D) NF-L filaments. The phosphorylation samples were taken at 120 min.
Fig.
then
BIOCHEMICAL
3, 1990
Edman degradation
under of
"Experimental these
with 1,2,3
37-53
phosphopeptides
the and and
reported 4 were l-14,
and
Vol.
167,
No.
3, 1990
A7
BIOCHEMICAL
Pi -
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B,
*
P-Ser -
AND
T ;” N
0
A =O.l
P-Thr P-Tyr -
TIME(Min)
Fig.
Table
1.
3. Phosphoamino acid analysis of phosphopeptide fragments phosphorylated by protein
Amino
acid
sequences
and for
Amino
Peptide
acid
30 33 Ser-Gly-Tyr-Ser-Thr-Ala-Arg
2
Val-His-Ile-Ser-Ser-Val-Arg
HPLC
of phosphorylation kinase C
of phosphopeptides
Relative
phosphateb
sequencea
1
23
sites protein
(A) and reverse-phase (B) of soluble NF-L kinase C for 120 min.
amount of (% of total)
36 10.3
----
27
29 23.7
----
37
Ser-Ala-Tyr-Ser-Ser-Tyr-Ser-Ala-Pro-Val-Ser51
Ser-Ser-Leu-m-Val-Arg
25.7
53
----
1
9
Ser-Ser-Phe-Ser-Tyr-Glu-Pro-Tyr-Tyr-Ser-Thr12
31.1
14
&-Tyr-Lys ---aResidue numbers above the sequences were determined from the aminoSolid and broken lines indicate terminal end of porcine NF-L (13). the phosphorylated serine and basic amino acids, respectively. bDetermined from radioactivity in the HPLC analysis, as shown in Fig. 3 yielded the radioactive 3. CChymotryptic digestion of peptide dPeptide 4 could not be peptide, Ser-Val-Arg (residues 51-53). Chymotryptic digestion yielded the sequenced from the amino-terminus. Tyr-Ser-Thr-Ser-Tyr-Lys (residues g-14). radioactive peptide, 1321
Vol.
167,
No.
BIOCHEMICAL
3, 1990
BIOPHYSICAL
RESEARCH
Rod
Head, 1
AND
Tail
I
58 92123137
399
COMMUNICATIONS
408
548
1 Head
SSFSYEPYYgT
8
YKRRYV *
4
EhRVHIS
8
-2--l--
VRiLY
g
YtfSYSAPVSSS?
9
TARSA
VRRSYSS
3 70 SSGLMPSLENLDLSQVAAISNDLKSIRTQEKAQLQDLNDR
90
110 FASFIERVHELEQQNKVLEAQLLVLRQKHSEPSRFRALYE
130
150 QEIRDLRLAAEDATNEKQALQGEREGLEETLRNLQARYEE
170
190 EVLSREDAEGRLMEARKGADEAALARAELEKRIDSLMDEl
210
230 AFLKKVHEEEIAELQAQIQYAQISVEMDVSSKPDLSAALK
250
270 DIRAQYEKLAAKNMQNAEEWFKSRFTVLTESAAKNTDAVR
290
310 AAKDEVSESRRLLKAKTLEIEACKGMNEALEKQLQELEDK
330
350 QNADISAMQDTINKLENELRTTKSENARYLKEYQDLLNVK
370
1 Rod
VTail
390 MALDIEIAAYRKLLEGEETRLSFTSVGSLTTGYSQSSQVF 430 GRSAYGGLQTSSYLMSTRSFPSYYTSHVQEZQIEVEETIE
450
470 AAKAEEAKDEPPSEGEAEEEGKEKEEAEAEAEAEEEGAQE
490
510 EEEAAEKEESEEAKEEEGGEGEQGEETKEAEEEEKKDEGA
530
GEEQAT'C
Fig.
K K D
4. Schematic structure of NF-L and the location of phosphorylation sites for protein kinase C. This scheme was based on the structural model of intermediate filament proteins proposed by Geisler and Weber (19). The isolated phosphopeptides underlined are aligned according to the sequence of ?+mino acids are represented by porcine NF-L (13). the single-letter code. Sites phosphorylated are shown by a P within a circle.
respectively. (residues the Fig.
Thus, l-548)
amino-terminal
for
all
the
protein head
domain
sites kinase
of
C are
(residues
4.
1322
phosphorylation
of
apparently l-58),
NF-L
located as
shown
at in
Vol.
167,
No.
BIOCHEMICAL
3, 1990
Phosphoamino kinase
acid
C revealed
phosphopeptides
analysis
contained
more
Table
1, we used
sites
of
phosphorylation. with
Meyer
(Table
et
residues
phenylthiohydantoin-serine
Only
the
fourth
cycle
of of
was
four
et
of is
amino-terminal and
been
reported
tail
domains
et
9-14),
methods
cycle of
normal and
of
the
peptide
2,
the
3 and the
peptide
4, that
Ser-51
(Table
1
the
and Ser-12.
the
amino-terminal
and
Fig.
around
the
NF-L
are
accord
with
al.
(15),
Woodgett
et
in
PTH-serine at
of
at
the
observed
indicating
located
serine
contrast,
peptide
of
of
protein
3). kinase
C
the al.
and
(16)
(17).
phosphorylation
domains
of
Ferrari
was
Ser-27,
lysine
sequences
the
In
thereby
are
Ser-Val-Arg
DTT adduct
fragment
or
primary
location
fifth
fragment
residues
al.
the
on Ser-33,
further
radioactive
(PTH-serine) PTH-serine.
shown),
arginine
of
Kishimoto
1,
not
sites
conclusions
the
the
located
to of
the
4 were
of
PTH-serine
chymotryptic
phosphorylation
The
peptide
serine
close
Features
of
(data
phosphate
side
DTT adduct
4 were
sequencing,
exclusively
chymotryptic
of
respectively
of
exact
(residues
gas-phase
adduct
provides
(14).
All
During
(DTT)
cycle
of
made use
the as
the
3 and
3 and
all
residues,
identify
sequences
We also
1).
were
cycle
to
peptides
(14).
phosphoserine
fourth
from
Since
serine
peptides
al.
dithiothreitol
first
two
methods
The
COMMUNICATIONS
by protein
3).
and Tyr-Ser-Thr-Ser-Tyr-Lys
51-53)
respectively
(Fig.
First,
derived
RESEARCH
phosphorylated
than
two
chymotrypsin.
phosphopeptides
BIOPHYSICAL
NF-L
phosphoserine
in
(residues
of
only
shown
digested
AND
the
domain
of
interest.
head
the
of
protein
Neurofilament
domains,
the
carboxyl-terminal that
has
kinase
tail
central
C
-helical
domains
on their 1323
potential
rod It
(18,lg).
phosphorylation-dephosphorylation no effect
contain
proteins
of to
polymerize
the
has
Vol.
167,
No.
3, 1990
BIOCHEMICAL
although
(201, seem
to
modulate
organelles kinase
these
types
of
Our
C phosphorylation modulates
between present of
the
the
head to
domain
neurofilament
proteins,
vimentin
(9,11,23-25),
(10,11,26,27)
and glial
fibrillary
acidic
Different
polymerization. possibly
be regulated
meurofilament
proteins
of
functions
neurofilament
proteins
of
domain
of desmin
protein in
the
(28), the
with
extent
neurofilaments
specific
by different
protein
Similar
and alternations
by the
that
polymerize.
made on family
phosphorylation
and other
indicate
have
domain
COMMUNICATIONS
neurofilaments
observations
head
RESEARCH
results
potential
been
BIOPHYSICAL
phosphorylation-dephosphorylation
interactions
(21,22).
protein
AND
of may
phosphorylation
protein
of
kinases.
Acknowledgments We are grateful to Drs. Y. Nishizuka and T. Takahashi for kind encouragement, to M. Ohara for pertinent comments and for secretarial services. This discussion and S. Tokumasu research was supported in part by a Grant-in-Aid for Scientific Research and a Grant for Cancer Research from the Ministry of Education, Science and Culture of Japan, and special coordination funds of the Science and Technology Agency of the Government of Japan.
References 1.
Schlaepfer, 78,
W.W.,
and Freeman,
L.A.
(1978)
J.
Cell
Biol.
653-662.
4.
Liem, R.K.H., Yen, S.H., Salomon, G.D., and Shelanski, M.L. (1978) J. Cell Biol. 79. 637-645. Anderton, B.H., Ayers, B.H., and Thorpe, R. (1978) FEBS 96, 159-l 63. Lett. Geisler, N., and Weber, K. (1981) J. Mol. Biol. 151, 565-
5.
Liem,
2. 3.
571.
R.K.H.,
and
R.K.,
Jeng,
Hutchison,
S.B.
(1982)
Biochemistry
21,
3221-3226. 6.
Sihag, 233,
7. 8. 9. 10.
A.Y.,
and Nixon,
R.A.
(1988)
FEBS Lett.
181-185.
Gonda, Y., Inagaki, M., Sato, C., Nishi, Y., Yoshida, T., and Yatani, R. (1988) Mie Med. J. 38, 343-356. Inagaki, M., Watanabe, M., and Hidaka, H. (1985) J. Biol. Chem. 260, 2922-2925. Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., and Sato, C. (1987) Nature 328, 649-652. Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 59705978. 1324
Vol.
167,
11. 12. 13. 14. 15. 16. 17.
18.
No.
20. 21. 22. 23. 24. 25. 26. 27. 28.
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Inagaki, M., Gonda, Y., Ando, S., Kitamura, S., Nishi, Y., and Sato, C. (1989) Cell Struct. Funct. 14, 279-286. Bidlingmeyer, B.A., Cohen, S.A., and Tarvin, T.L. (1984) J. Chromatogr. 336, 93-104. Geisler, N., Plessmann, U., and Weber, K. (1985) FEBS Lett. 182, 475-478. Meyer, H.E., Hoffmann-Posorske, E., Korte, H., and Heilmeyer, L.M.G.Jr. (1986) FEBS Lett. 204, 61-66. Ferrari, S., Marchiori, F., Borin, G., and Pinna, L.A. (1985) FEBS Lett. 184, 72-77. Woodgett, J.R., Gould, K.L., and Hunter, T. (1986) Eur. J. Biochem. 161, 177-184. Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuiji, Y ., Nomura, H., Takeyama, Y., and Nishizuka, Y. (1985) J. Biol. Chem. 260, 12492-12499. Steinert, P.M., and Roop, D.R. (1988) Ann Rev. Biochem. 57,
19.
3, 1990
593-625.
in Cell and Molecular Geisler, N., and Weber, K. (1986) Biology of the Cytoskeleton (Shay, J.W., Ed.) ~~41-68, Plenum Press, New York. Georges, E., Lefebvre, S., and Mushynski, W.E. (1986) J. Neurochem. 47, 477-483. J. Cell Biol. 94, 129-142. Hirokawa, N. (1982) Hirokawa, N., Glicksman, M.A., and Willard, M.B. (1984) J. Cell Biol. 98, 1523-1536. FEBS Lett. 234, 73-78. Evans, R.M. (1988) Ando, S., Tanabe, K., Gonda, Y., Sato, C., and Inagaki, M. (1989) Biochemistry 28, 2974-2979. Inagaki, M., Takahara, H., Nishi, Y., Sugawara, K., and J. Biol. Chem. 264, 18119-18127. Sato, C. (1989) Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20. Kitamura, S., Ando, S., Shibata, M., Tanabe, K., Sato, C., and Inagaki, M. (1989) J. Biol. Chem. 264, 5674-5678. Inagaki, M., Gonda, Y., Nishizawa, K., Kitamura, S., Sato, C ., Ando, S., Tanabe, K., Kikuchi, K., Tsuiki, S., and Nishi, Y. J. Biol. Chem. in press.
1325
167,
March
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
RESEARCH
Pages
1990
1316-1325
INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF ASSEMBLYDISASSEMBLY OF NEUROFILAMENTS IN VITRO Yasunori
Gonda+, + Kitamura+,
Shinobu
Kimiko
Nishizawa,
Yasushi
Minouran,
and Laboratory
of Experimental
Aichi
§Life
Masaki
Cancer
Center
Information
Inagaki
Radiology Research
Shoji
Ando',
Yoshimi
Nishi§,
* and qBiophysics
Institute,
Nagoya
464, Japan
Analysis
Center,
Unit,
Chikusa-ku,
Komaki 485,
Japan
Received February 15, 1990 Protein kinase C phosphorylated the major mammalian neurofilament protein (NF-L) with ~3 mol phosphate per mol protein. The phosphorylated NF-L no longer formed the filaments. Sequential analysis of the tryptic phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-27, Ser-33 and Ser-51 were phosphorylated by protein kinase C. These findings contribute toward elucidation of mechanisms 01990 Academic Press, Inc. regulating the functions of neurofilaments.
Neurofilaments
are
neuronal
cytoskeleton
neuronal
size
major
and serve
and shape.
electrophoresis
showed that
polypeptides
with
160,000
(NF-M),
and 68,000
as
filaments
Life Science Research Co., LTD., Samejima,
*To whom correspondence
and Biological should
0 1990 by Academic Press, Inc. in any form reserved.
of reproducrion
1316
200,000
and the
of
gel consist (NF-H),
In reconstitution two high-
Laboratories, Asahi Fuji 416, Japan. Laboratories,
be addressed.
0006-291X/90$1.50
the
determinants
sulfate
masses of
(NF-L)(l-5).
only
$Present address: Medical Nagano 396, Japan.
Copyright AI1 rights
important
of
mammalian neurofilaments
molecular
NF-L formed
+Present address: Chemical Industry
components
Sodium dodecyl
three
experiments,
structural
Ina,
of
Vol.
167,
No.
3, 1990
molecular
weight
self-assembly,
proteins although
filaments core
BIOCHEMICAL
formed
of
the
Sihag
et
can
serve
vivo
(6).
al. as
both
by NF-L
that
a substrate
for
is
significance the
were
RESEARCH
showed
readily Thus,
action shows
of of
that
the
COMMUNICATIONS
evidence
incorporated NF-L
of into
may be the
filament-forming
protein
a paucity
the
central
kinase
in
protein
the
C, in
data
kinase
protein, vitro
related
kinase
C on NF-L.
protein
kinase
C stoichiometrically
NF-L
and that
kinase
phosphorylation
functional of
Experimental
the
NF-L and
to
in
the
C phosphorylation,
protein
phosphorylates follows
and NF-M)
(4,5).
reported
As there
we obtained
(NF-H
BIOPHYSICAL
filaments.
functional examined
AND
alteration specific
we
The evidence
by this domain.
Procedures
Purification of proteinsNF-L was purified from porcine brain white matter by the method of Liem and Hutchison (5) with some modifications (7). Protein kinase C from the rat brain was prepared by the method of Inagaki et al. (8). Phosphorylation of NF-LNF-L (1 mg/ml) was dialyzed against 5 mM Tris-HCl, pH 8.8, 2mM EGTA, 1 mM PMSF and 50 mM 2mercaptoethanol (Buffer A) for 24hr at 4OC for phosphorylation of the soluble form of NF-L, or against 10 mM PIPES, pH 7.0, 0.15 M NaCl, 2 mM EGTA, 1 mM PMSF and 50 mM 2-mercaptoethanol (Buffer B) for 6 hr at 37OC for phosphorylation of the filamentous form of NF-L. Both forms of NF-L (0.15 mg/ml) were ted by incubation with 5 ug/ml of protein kinase C, ~h~s~~o~?32p-~Tp 0.3 mM MgC12, 25 mM NaCl and 10 mM PIPES (pH 7:01
(9-11).
’
Electron microscopyIntermediate filament preparations were placed directly on carbon film-coated specimen grids and stained with 1 % or 2 % uranyl acetate or 2 % sodium phosphotungstate (pH 7.2). Phosphopeptides from35 adioactive NF-LSoluble NF-L (2 mg) was incubated with [vP] ATP and protein kinase C (40 pg) in an 8-ml mixture, as described above. The phosphorylated NF-L was precipitated with 10 % trichloroacetic acid and collected by centrifugation for 30 min at 10,000 x g at 4OC. The precipitated NF-L was dissolved in 7 M urea (3 ml), and dialysed overnight against 10 mM Tris-HCl (pH 8.6) at 4 OC. To the sample solution, we added 400 1-11 of 250 mM Tris-HCl (pH 7.5) and the final protein concentration was adjusted to 0.5 The phosphorylated NF-L mg/ml with 25 mM Tris-HCl (pH 7.5). (0.5 mg/ml) in 25 mM Tris-HCl (pH 7.5) was treated with L-ltosylamide-2-phenylethyl chloromethyl ketone-treated trypsin A 2 ml sample of l/25 (w/w) of NF-L) at 37OC for 2 h. (Sigma, the reaction mixture was applied to a Zorbax C8 (0.46 x 20 cm) column attached to a Waters HPLC system (model 510 pumps, a model 490 detector and an automated gradient controller) and 1317
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was
eluted with a linear gradient of IO-50 % acetonitrile in % trifluoroacetic acid over 70 min followed by a further linear gradient of 50-80 % acetonitrile in 0.1 % trifluoroacetic acid over 20 min at a flow rate of 0.8 ml/min. Elution of the fragments was monitored by UV at 230 nm and by the radioactivity of each fraction (0.8 ml). Purification of fragmentsThe radioactive fractions were applied to an anion exchange column of TSK gel QAE-2SW (0.46 x 25 cm) and were eluted with a linear gradient of O-O.5 M NaCl in 20 mM Tris-HCl (pH 7.5) over 40 min at a flow rate of 0.8 mllmin. Each radioactive fraction obtained was then applied to a Zorbax C8 (0.46 x 20 cm) column and was eluted with the same linear gradient used in the first fractionation of the at the same flow rate. Radioactive fractions phosphopeptides, obtained were lyophilized and stored at 4OC. Phosphoamino acid analysisProtein kinase C phosphorylated NF-L was subjected to acid hydrolysis in 6 N HCl for 1.5 hr at llo"c. The phosphoamino acids were resolved by electrophoresis at pH 3.5 on a cellulose thin layer plate, as described (9). Amino acid analysisPurified phosphopeptides (0.5-I nmol) were subjected to 6 N HCl hydrolysis in vacua at 110 OC for 24 hr. Amino acid analysis was performed by reverse-phase HPLC of phenylthiocarbamoyl derivatives (12) using the Waters Pica-Tag system. Sequence analysisA I%7 n mol sample of the purified fragments dissolved in 0.1 % trifluoroacetic acid was analyzed using an ABI 470A gas-phase sequencer equipped with an ABI 120A on-line PTH amino acid analyzer, using the 03R PTH program. 0.1
Results Protein of
kinase
NF-L.C of
shown
in
protein the
IA.
incorporated Protein
protein
kinase
a more
of
NF-L
NF-L
over
4 hr.
was
mol
of Thus,
the
for
protein
phosphorylated
competence
of
the
was
The amount
of
polymerized
with
increases
in
the
phosphate
protein
in 1318
the
as
used
phosphate
filamentous
kinase
NF-L
kinase NF-L (Fig.
1 mol
of
the
was
end of
soluble
is substrate
was
4 hr. form
form
of
of
NF-L
NF-
was
C.
by protein
polymerization analyzed.
as
into
the the
NF-L
phosphate
of
molecule'at
substrate
NF-L
of
of
used
filament
C, 21
by protein forms
was
~3 mol
NF-L
assembly-disassembly
phosphorylation
C phosphorylation for
the
filamentous
kinase
mol
appropriate soluble
and
When the
per
L continued
of
C incorporated
for
and
When soluble
molecule.
substrate
course
soluble
kinase
NF-L
The
time
both
Fig.
Discussion
C phosphorylation
The
kinase
and
kinase C-treated
C and NF-L
(ppt)
decreased
IB).
The
same
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B, soluble NF-L SP
SP
5
Tii
Fig
set
of
of
NF-L.
1.
finding
The
of
90
5
60
15 30 The(min)
is
in
good
agreement
of
in
2. C acts
the
of
These as
dissolved
for NF-L
120
90
with
NF-L
the
results
the
filament
a regulator
from
slow by
phosphorylated are
the
240
120
140
NF-L
rate
NF-L
form
filament
(Fig.
protein
interpreted of
filamentous
manner
in a time-dependent
slowly,
findings
in
30 60 Time(min)
NF-L
was performed
amount
Typical
kinase
SPSPSP
C, filamentous
(nlln)
experiments
phosphorylation
Fig.
15
SP
Time course of phosphorylation of NF-L by protein kinase C (A) and effect of phosphorylation on polymerization competence of soluble NF-L (B) and on the state of filamentous NF-L (C). A; phosphorylation of soluble and filamentous NF-L is represented by open circles and closed circles, 30, 60, 90, 120 and respectively. B; after 0, 5, 15, 240 min incubation with protein kinase C, this assay mixture was passed through a Millipore filter (0.22 to remove vesicles of phosphatidylserine and was pm) then dialyzed against Buffer B containing 7 M urea polymerization for 12 hr at 4OC and then against Buffer B for 6 hr at 37OC to promote filament formation. After centrifugation at 100,000 x g for 30 min, supernatants (s) and precipitates (p) were subjected to 10 % SDS-PAGE. C; after 0, 5, 15, 30, 60, 90, 120 and 240 min incubation with protein kinase C, the reaction mixtures were directly centrifuged at 100,OOOxg and then supernatants (s) and precipitates (p) were subjected to 10 % SDS-PAGE.
the
increased
SPSP
c).
1
of kinase
samples to
This
mean
C. are
shown
that
assembly-disassembly
protein of
vitro. Phosphorylation
identify
the
sites
site
located
on
phosphorylated
the
head
by protein 1319
domain-
kinase
To
C,
NF-L,
Vol.
167,
No.
radioactive
As
NF-L
subjected shown
in
Fig.
digested
3,
the
acid
COMMUNICATIONS
The in of at
Table
porcine NF-L
for
1.
NF-L
residues
acid In
purified
by anion
a comparison
(13),
peptides
30-36,
23-29,
exchange
under
radioactive
gas-phase
sequences
was
several
as described
1320
material column.
separated purified
as described
amino
and the
a reverse-phase
further
Each
analyzed
compositions,
listed
with
HPLC columns,
was
Procedures".
located
was
Procedures".
phosphopeptide
sequence
RESEARCH
trypsin
HPLC procedure
each
re-reverse-phase
amino
BIOPHYSICAL
with
HPLC equipped
and
"Experimental
are
was to
phosphopeptides and
AND
2. Electron microscopy of the negatively stained NF-L samples. A,B; polymerization competence of nonphosphorylated (A) and protein kinase C C,D; the state of phosphorylated (B) soluble NF-L. non-phosphorylated (C) and protein kinase C phosphorylated (D) NF-L filaments. The phosphorylation samples were taken at 120 min.
Fig.
then
BIOCHEMICAL
3, 1990
Edman degradation
under of
"Experimental these
with 1,2,3
37-53
phosphopeptides
the and and
reported 4 were l-14,
and
Vol.
167,
No.
3, 1990
A7
BIOCHEMICAL
Pi -
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B,
*
P-Ser -
AND
T ;” N
0
A =O.l
P-Thr P-Tyr -
TIME(Min)
Fig.
Table
1.
3. Phosphoamino acid analysis of phosphopeptide fragments phosphorylated by protein
Amino
acid
sequences
and for
Amino
Peptide
acid
30 33 Ser-Gly-Tyr-Ser-Thr-Ala-Arg
2
Val-His-Ile-Ser-Ser-Val-Arg
HPLC
of phosphorylation kinase C
of phosphopeptides
Relative
phosphateb
sequencea
1
23
sites protein
(A) and reverse-phase (B) of soluble NF-L kinase C for 120 min.
amount of (% of total)
36 10.3
----
27
29 23.7
----
37
Ser-Ala-Tyr-Ser-Ser-Tyr-Ser-Ala-Pro-Val-Ser51
Ser-Ser-Leu-m-Val-Arg
25.7
53
----
1
9
Ser-Ser-Phe-Ser-Tyr-Glu-Pro-Tyr-Tyr-Ser-Thr12
31.1
14
&-Tyr-Lys ---aResidue numbers above the sequences were determined from the aminoSolid and broken lines indicate terminal end of porcine NF-L (13). the phosphorylated serine and basic amino acids, respectively. bDetermined from radioactivity in the HPLC analysis, as shown in Fig. 3 yielded the radioactive 3. CChymotryptic digestion of peptide dPeptide 4 could not be peptide, Ser-Val-Arg (residues 51-53). Chymotryptic digestion yielded the sequenced from the amino-terminus. Tyr-Ser-Thr-Ser-Tyr-Lys (residues g-14). radioactive peptide, 1321
Vol.
167,
No.
BIOCHEMICAL
3, 1990
BIOPHYSICAL
RESEARCH
Rod
Head, 1
AND
Tail
I
58 92123137
399
COMMUNICATIONS
408
548
1 Head
SSFSYEPYYgT
8
YKRRYV *
4
EhRVHIS
8
-2--l--
VRiLY
g
YtfSYSAPVSSS?
9
TARSA
VRRSYSS
3 70 SSGLMPSLENLDLSQVAAISNDLKSIRTQEKAQLQDLNDR
90
110 FASFIERVHELEQQNKVLEAQLLVLRQKHSEPSRFRALYE
130
150 QEIRDLRLAAEDATNEKQALQGEREGLEETLRNLQARYEE
170
190 EVLSREDAEGRLMEARKGADEAALARAELEKRIDSLMDEl
210
230 AFLKKVHEEEIAELQAQIQYAQISVEMDVSSKPDLSAALK
250
270 DIRAQYEKLAAKNMQNAEEWFKSRFTVLTESAAKNTDAVR
290
310 AAKDEVSESRRLLKAKTLEIEACKGMNEALEKQLQELEDK
330
350 QNADISAMQDTINKLENELRTTKSENARYLKEYQDLLNVK
370
1 Rod
VTail
390 MALDIEIAAYRKLLEGEETRLSFTSVGSLTTGYSQSSQVF 430 GRSAYGGLQTSSYLMSTRSFPSYYTSHVQEZQIEVEETIE
450
470 AAKAEEAKDEPPSEGEAEEEGKEKEEAEAEAEAEEEGAQE
490
510 EEEAAEKEESEEAKEEEGGEGEQGEETKEAEEEEKKDEGA
530
GEEQAT'C
Fig.
K K D
4. Schematic structure of NF-L and the location of phosphorylation sites for protein kinase C. This scheme was based on the structural model of intermediate filament proteins proposed by Geisler and Weber (19). The isolated phosphopeptides underlined are aligned according to the sequence of ?+mino acids are represented by porcine NF-L (13). the single-letter code. Sites phosphorylated are shown by a P within a circle.
respectively. (residues the Fig.
Thus, l-548)
amino-terminal
for
all
the
protein head
domain
sites kinase
of
C are
(residues
4.
1322
phosphorylation
of
apparently l-58),
NF-L
located as
shown
at in
Vol.
167,
No.
BIOCHEMICAL
3, 1990
Phosphoamino kinase
acid
C revealed
phosphopeptides
analysis
contained
more
Table
1, we used
sites
of
phosphorylation. with
Meyer
(Table
et
residues
phenylthiohydantoin-serine
Only
the
fourth
cycle
of of
was
four
et
of is
amino-terminal and
been
reported
tail
domains
et
9-14),
methods
cycle of
normal and
of
the
peptide
2,
the
3 and the
peptide
4, that
Ser-51
(Table
1
the
and Ser-12.
the
amino-terminal
and
Fig.
around
the
NF-L
are
accord
with
al.
(15),
Woodgett
et
in
PTH-serine at
of
at
the
observed
indicating
located
serine
contrast,
peptide
of
of
protein
3). kinase
C
the al.
and
(16)
(17).
phosphorylation
domains
of
Ferrari
was
Ser-27,
lysine
sequences
the
In
thereby
are
Ser-Val-Arg
DTT adduct
fragment
or
primary
location
fifth
fragment
residues
al.
the
on Ser-33,
further
radioactive
(PTH-serine) PTH-serine.
shown),
arginine
of
Kishimoto
1,
not
sites
conclusions
the
the
located
to of
the
4 were
of
PTH-serine
chymotryptic
phosphorylation
The
peptide
serine
close
Features
of
(data
phosphate
side
DTT adduct
4 were
sequencing,
exclusively
chymotryptic
of
respectively
of
exact
(residues
gas-phase
adduct
provides
(14).
All
During
(DTT)
cycle
of
made use
the as
the
3 and
3 and
all
residues,
identify
sequences
We also
1).
were
cycle
to
peptides
(14).
phosphoserine
fourth
from
Since
serine
peptides
al.
dithiothreitol
first
two
methods
The
COMMUNICATIONS
by protein
3).
and Tyr-Ser-Thr-Ser-Tyr-Lys
51-53)
respectively
(Fig.
First,
derived
RESEARCH
phosphorylated
than
two
chymotrypsin.
phosphopeptides
BIOPHYSICAL
NF-L
phosphoserine
in
(residues
of
only
shown
digested
AND
the
domain
of
interest.
head
the
of
protein
Neurofilament
domains,
the
carboxyl-terminal that
has
kinase
tail
central
C
-helical
domains
on their 1323
potential
rod It
(18,lg).
phosphorylation-dephosphorylation no effect
contain
proteins
of to
polymerize
the
has
Vol.
167,
No.
3, 1990
BIOCHEMICAL
although
(201, seem
to
modulate
organelles kinase
these
types
of
Our
C phosphorylation modulates
between present of
the
the
head to
domain
neurofilament
proteins,
vimentin
(9,11,23-25),
(10,11,26,27)
and glial
fibrillary
acidic
Different
polymerization. possibly
be regulated
meurofilament
proteins
of
functions
neurofilament
proteins
of
domain
of desmin
protein in
the
(28), the
with
extent
neurofilaments
specific
by different
protein
Similar
and alternations
by the
that
polymerize.
made on family
phosphorylation
and other
indicate
have
domain
COMMUNICATIONS
neurofilaments
observations
head
RESEARCH
results
potential
been
BIOPHYSICAL
phosphorylation-dephosphorylation
interactions
(21,22).
protein
AND
of may
phosphorylation
protein
of
kinases.
Acknowledgments We are grateful to Drs. Y. Nishizuka and T. Takahashi for kind encouragement, to M. Ohara for pertinent comments and for secretarial services. This discussion and S. Tokumasu research was supported in part by a Grant-in-Aid for Scientific Research and a Grant for Cancer Research from the Ministry of Education, Science and Culture of Japan, and special coordination funds of the Science and Technology Agency of the Government of Japan.
References 1.
Schlaepfer, 78,
W.W.,
and Freeman,
L.A.
(1978)
J.
Cell
Biol.
653-662.
4.
Liem, R.K.H., Yen, S.H., Salomon, G.D., and Shelanski, M.L. (1978) J. Cell Biol. 79. 637-645. Anderton, B.H., Ayers, B.H., and Thorpe, R. (1978) FEBS 96, 159-l 63. Lett. Geisler, N., and Weber, K. (1981) J. Mol. Biol. 151, 565-
5.
Liem,
2. 3.
571.
R.K.H.,
and
R.K.,
Jeng,
Hutchison,
S.B.
(1982)
Biochemistry
21,
3221-3226. 6.
Sihag, 233,
7. 8. 9. 10.
A.Y.,
and Nixon,
R.A.
(1988)
FEBS Lett.
181-185.
Gonda, Y., Inagaki, M., Sato, C., Nishi, Y., Yoshida, T., and Yatani, R. (1988) Mie Med. J. 38, 343-356. Inagaki, M., Watanabe, M., and Hidaka, H. (1985) J. Biol. Chem. 260, 2922-2925. Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., and Sato, C. (1987) Nature 328, 649-652. Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 59705978. 1324
Vol.
167,
11. 12. 13. 14. 15. 16. 17.
18.
No.
20. 21. 22. 23. 24. 25. 26. 27. 28.
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Inagaki, M., Gonda, Y., Ando, S., Kitamura, S., Nishi, Y., and Sato, C. (1989) Cell Struct. Funct. 14, 279-286. Bidlingmeyer, B.A., Cohen, S.A., and Tarvin, T.L. (1984) J. Chromatogr. 336, 93-104. Geisler, N., Plessmann, U., and Weber, K. (1985) FEBS Lett. 182, 475-478. Meyer, H.E., Hoffmann-Posorske, E., Korte, H., and Heilmeyer, L.M.G.Jr. (1986) FEBS Lett. 204, 61-66. Ferrari, S., Marchiori, F., Borin, G., and Pinna, L.A. (1985) FEBS Lett. 184, 72-77. Woodgett, J.R., Gould, K.L., and Hunter, T. (1986) Eur. J. Biochem. 161, 177-184. Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuiji, Y ., Nomura, H., Takeyama, Y., and Nishizuka, Y. (1985) J. Biol. Chem. 260, 12492-12499. Steinert, P.M., and Roop, D.R. (1988) Ann Rev. Biochem. 57,
19.
3, 1990
593-625.
in Cell and Molecular Geisler, N., and Weber, K. (1986) Biology of the Cytoskeleton (Shay, J.W., Ed.) ~~41-68, Plenum Press, New York. Georges, E., Lefebvre, S., and Mushynski, W.E. (1986) J. Neurochem. 47, 477-483. J. Cell Biol. 94, 129-142. Hirokawa, N. (1982) Hirokawa, N., Glicksman, M.A., and Willard, M.B. (1984) J. Cell Biol. 98, 1523-1536. FEBS Lett. 234, 73-78. Evans, R.M. (1988) Ando, S., Tanabe, K., Gonda, Y., Sato, C., and Inagaki, M. (1989) Biochemistry 28, 2974-2979. Inagaki, M., Takahara, H., Nishi, Y., Sugawara, K., and J. Biol. Chem. 264, 18119-18127. Sato, C. (1989) Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20. Kitamura, S., Ando, S., Shibata, M., Tanabe, K., Sato, C., and Inagaki, M. (1989) J. Biol. Chem. 264, 5674-5678. Inagaki, M., Gonda, Y., Nishizawa, K., Kitamura, S., Sato, C ., Ando, S., Tanabe, K., Kikuchi, K., Tsuiki, S., and Nishi, Y. J. Biol. Chem. in press.
1325
