From the Department of Pediatrics and the
§ Departments of Pathology and Molecular, Cellular, and
Developmental Biology, Yale University,
New Haven, Connecticut 06510
Received for publication, April 23, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Spectrin is a ubiquitous heterodimeric
scaffolding protein that stabilizes membranes and organizes protein and
lipid microdomains on both the plasma membrane and intracellular
organelles. Phosphorylation of Post-translational protein modification regulates cellular
function. Although in many instances the role of such modification is
well understood, less clear is the impact of protein modification on
cytoskeletal dynamics. This is particularly true for spectrin, a large
multifunctional scaffolding molecule positioned at the interface
between membrane and cytosol. Produced by seven distinct genes, the
spectrin family segregates as two subunits, Several pathways of post-translational regulation impact spectrin. The
Other regulatory pathways impacting spectrin include the action of
calcium, calmodulin, calcium-activated proteolysis, and the regulation
of its Golgi binding by ARF1, a small GTPase. ARF1 acts by modifying
phosphatidylinositol 4,5-bisphosphate levels in Golgi membranes, a
substrate for the pleckstrin homology domain of In the present study we demonstrate that In Vivo Determination of Maitotoxin Induced Activation of Calpain Resulting in c-Src Binding Assays--
Confluent MDCK cells were treated with
pervanadate as above at 37 °C and solubilized in binding buffer (20 mM HEPES (pH 7.5), 25 mM KCl, 120 mM NaCl, 2 mM EGTA, 2 mM EDTA, 0.2 mM DTT, 0.5% Triton X 100, and 0.1 mM PMSF).
This lysate was incubated with GST fusion peptides representing the SH3
domains of Recombinant Spectrin Peptides and in Vitro Tyrosine
Phosphorylation--
Oligonucleotides were designed so as to amplify
from human Calpain Cleavage Assays--
Recombinant GST fusion peptides of
spectrin were incubated with µ-calpain (Calbiochem) (E/S = 1/600
M/M) for 15 min at 30 °C in a total reaction
volume of 25 µl. Reaction conditions included 600 µM
CaCl2, 20 mM Tris-HCl, pH 7.5, 25 mM NaCl, and 10 mM DTT. When phosphorylated
peptides were required, they were prepared by pre-incubation of the
recombinant peptide with c-Src (E/S = 1:100 to 1:1000
M/M) under conditions as above. The level of
phosphorylation was monitored by incorporated 32P or by
Western blotting with anti-phosphotyrosine antibody clone G10 (Upstate Biotechnology).
Other Procedures--
Protein determinations used the Bradford
assay (Calbiochem). SDS-PAGE analysis utilized gradient Laemmli
polyacrylamide gels. Molecular biology procedures followed standard
methods (37).
Tyrosine Phosphorylation of
Although in most experiments the level of
Finally, it is interesting to note that when there is immediate harvest
of untreated confluent MDCK cells into a SDS buffer, with care taken to
block postextraction proteolysis by the early and liberal use of
effective protease inhibitors, one achieves cell lysates that are
nearly devoid of spontaneous c-Src Binds to the SH3 Domain of Recombinant
To identify the specific sites of tyrosine phosphorylation in SACC, a
set of smaller recombinant peptides were examined that individually
represented each of the structural and functional motifs in SACC (Fig.
5A). These peptides were
incubated in vitro with c-Src, and pTyr was measured by
immunoblotting with anti-phosphotyrosine antibody (Fig. 5B).
Both the SA and S+ peptides (representing
Examination of the recombinant peptides CC and C (encompassing the
calpain cleavage site and calmodulin-binding domain in repeat 11 of
Phosphorylation of Tyr1176 Retards The studies presented here establish that Calpain cleavage of proteins is important in many cellular and
pathologic processes such as long-term potentiation and synaptic remodeling, glutamate-induced neurotoxicity, ischemic cellular injury,
apoptosis, platelet activation, exocrine secretion, neutrophil activation, mitosis, progesterone and estrogen receptor modulation, and
the regulation of a variety of kinases such as protein kinase C,
phosphorylase kinase, myosin light chain kinase,
calmodulin-dependent kinase and phosphatase, and other
signal transduction pathways (for reviews, see Refs. 33, 42, 48-51).
As cytoskeletal proteins are common substrates for
calcium-dependent proteases, regulating the degree of
calpain cleavage through tyrosine phosphorylation of the substrate
represents an important mechanism for modulating these cellular and
pathologic events. While it is likely that the novel pathway for the
control of spectrin breakdown reported here will be utilized in a
variety of physiologic and pathologic settings, its role in neuronal
function may be particularly important. Spectrin and other
cytoskeletal-associated proteins interact with the glutamate-gated
N-methyl-D-aspartate receptor (NMDA receptor) (for review, see Ref. 52). NMDA receptors, a class of glutamate-gated cation channels with high Ca2+ conductance, mediate fast
transmission and plasticity of central nervous system excitatory
synapses. Spectrin associates with the NR2 cytosolic subunit of the
NMDA receptor (19), an activity regulated by phosphorylation of the NR2
subunit. Calpain proteolysis of NR2 disrupts its association with
spectrin, conversely its phosphorylation by c-Src protects it from
calpain cleavage (36). Our present results suggest a related pathway by
which tyrosine phosphorylation of -spectrin on Ser/Thr is well
recognized. Less clear is whether
-spectrin is phosphorylated
in vivo and whether spectrin is phosphorylated on tyrosine
(pTyr). We affirmatively answer both questions. In cultured
Madin-Darby canine kidney cells,
II spectrin undergoes in vivo tyrosine phosphorylation. Enhancement of the steady
state level of pTyr-modified
II spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between
II spectrin
phosphorylation and dephosphorylation. Recombinant peptides containing
the Src homology 3 domain of
II spectrin (but not the Src homology 3 domain of
I spectrin) bind specifically to phosphorylated c-Src in
Madin-Darby canine kidney cell lysates, suggesting that this kinase is
responsible for its in vivo phosphorylation. pTyr-modified
II spectrin is resistant to maitotoxin-induced cleavage by
µ-calpain in vivo. In vitro studies of
recombinant
II spectrin peptides representing repeats 9-12 identify
two sites of pTyr modification. The first site is at
Tyr1073, a residue immediately adjacent to a region
encoded by alternative exon usage (insert 1). The second site is at
Tyr1176. This residue flanks the major site of cleavage by
the calcium-dependent protease calpain, and phosphorylation
of Tyr1176 by c-Src reduces the susceptibility of
II
spectrin to cleavage by µ-calpain. Calpain cleavage of spectrin,
activated by Ca2+ and calmodulin, contributes to diverse
cellular processes including synaptic remodeling, receptor-mediated
endocytosis, apoptosis, and the response of the renal epithelial cell
to ischemic injury. Tyrosine phosphorylation of
II spectrin now
would appear to also mediate these events. The spectrin skeleton thus
forms a point of convergence between kinase/phosphatase and
Ca2+-mediated signaling cascades.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
. Heterodimeric
II
II spectrin is the most common, expressed in most if not all
vertebrate cells. By binding to an array of integral membrane and
cytosolic proteins and to acidic phospholipids, spectrin links membrane
protein and lipid microdomains to the actin and microtubule filamentous
skeleton (for a review, see Ref. 1).
I,
II, and
III isoforms are phosphorylated on Ser and Thr
(2-5); spectrins
IV (6) and
V (7) may also be similarly
phosphorylated, although no data yet exists for these proteins. The
functions ascribed to
-spectrin phosphorylation include
destabilization of the erythrocyte membrane skeleton (8-10), disassembly of the skeleton during mitosis (4), and the control of
Golgi stability (11). However, the mechanism of these effects remains
elusive. Less clear is whether spectrin can be tyrosine-phosphorylated and whether the
-subunit of spectrin is ever covalently
phosphorylated. One report indicates that
-spectrin can be
tyrosine-phosphorylated when incubated in vitro with
purified insulin receptor kinase (12). Another report has appeared
indicating that both spectrin subunits are tyrosine-phosphorylated when
incubated in vitro with a spleen protein tyrosine kinase
(13). A recent report, appearing after the present study was first
submitted for publication, indicates that
II spectrin is
tyrosine-phosphorylated in cultured COS-7 cells in vivo
(14).
I
2 and
III
spectrin (15). Calcium binds directly to two EF-hand domains near the
COOH terminus of
-spectrin (16) with unknown functional
consequences. Calmodulin binds to a non-homologous sequence inserted
into the 11th repeat unit of vertebrate
II spectrin (17). Binding at
this site modifies the susceptibility of the nearby
Tyr1176-Gly1177 bond to cleavage by µ-calpain
(18) and renders the adjacent
II spectrin subunit susceptible
to µ-calpain cleavage at
Gln1441-Ser1442
(18).1 After calpain
cleavage, spectrin's self-association, actin binding, and membrane
binding properties are modified (20, 21). Spectrin proteolysis by
calpain has been correlated with processes involved with secretion and
endocytosis in epithelial cells (22-24), opacification of the
vertebrate lens (25), synaptic plasticity and long-term potentiation in
the central nervous system (26-28), and various central nervous
system pathologies including neurotoxic or ischemic injury
(e.g. see Refs. 29-33).
II spectrin is subject to
tyrosine phosphorylation in vivo in cultured Madin-Darby canine kidney (MDCK)2 cells
and that such phosphorylation bestows on
II spectrin resistance to
the calpain-mediated proteolysis that follows maitotoxin exposure. In vitro studies document at least two sites of tyrosine
phosphorylation in
II spectrin. One is at the site of calpain
cleavage, the other flanks a short sequence encoded by alternative exon
utilization. Both sites are adjacent to the SH3 domain of spectrin, a
locus that we show binds specifically to c-Src in MDCK cell extracts. Collectively, these data together with a recent independent report that
appeared after this study was first submitted (14) establish a role for
tyrosine phosphorylation of
II spectrin as a modifier of the calpain
sensitivity of spectrin and reveal the spectrin cortical skeleton as a
point of convergence between tyrosine kinase/phosphatase and
Ca2+-mediated signal transduction pathways.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
II Spectrin Tyrosine
Phosphorylation--
MDCK cells were cultured to confluence with
Dulbecco's modified Eagle's medium (DMEM) as before (34). In some
experiments when blockage of endogenous phosphatase was desired, cells
were washed three times with serum-free DMEM and exposed to 0.1 mM pervanadate (freshly prepared from aliquots of 100 mM activated sodium orthovanadate (Sigma) dissolved in
water and 100 mM H2O2) in Opti-MEM
I at 37 °C for 30 min. Control cells were incubated in Opti-MEM I
only. For immunoprecipitation, pervanadate-treated and -untreated MDCK
cells were washed three times in ice-cold PBS, lysed gently at 4 °C
for 15 min with 1 ml of modified RIPA lysis buffer (Tris-HCl 50 mM, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate,
150 mM NaCl, 1 mM PMSF, 1 mg/ml each of
aprotinin and leupeptin in a 1:100 dilution of Calbiochem phosphatase
inhibitor mixture set II (catalog number 524625). After centrifugation
at 14,000 rpm for 15 min at 4 °C, supernatants were analyzed by
SDS-PAGE or incubated (0.5 ml) with 3 µl of antibody overnight at
4 °C. Immune complexes were captured with 25 µl of packed
ImmunoPure® Plus immobilized protein G beads (Pierce) (2 h, 4 °C),
washed four times with modified RIPA lysis buffer, and analyzed by
SDS-PAGE. For Western blotting, proteins transferred to nitrocellulose
membranes were blocked for 1 h with either 5% (w/v) dry skim milk
in TBST buffer (20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, and 0.1% Tween 20) or in blocking buffer (1 M
glycine, 5% (w/v) dry skim milk, 1% (w/v) bovine serum albumin, and
5% (v/v) bovine calf serum). Proteins were detected with primary
antibodies in TBST at room temperature for 1 h at a typical
dilution of 1:1000 or as specified. The anti-glutathione S-transferase (GST) antibody (Amersham Biosciences)
was used at 1:2000. Detection employed goat
anti-rabbit/anti-mouse conjugated horseradish peroxidase at 1:10,000
and enhanced chemiluminescence (ECL, Amersham Biosciences).
II
Spectrin Proteolysis from Vanadate-pretreated MDCK Cells--
MDCK
cells were grown to confluence as above and treated with 5 mM activated sodium orthovanadate in Opti-MEM I without
H2O2. Cells were then washed with Opti-MEM I
one time, and 100 nM maitotoxin (Calbiochem) diluted in
Opti-MEM I was applied to cells for various periods at 37 °C. Cells
were harvested by washing one time in ice-cold PBS then scraped
immediately thereafter into 250 µl of one time sample (4% SDS, 60 mM Tris, pH 6.8, 10% v/v glycerol, 1 mM PMSF,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.2 mM DTT, 10 µg/ml protease arrest (Calbiochem), 1 µg/ml pepstatin, 1 mM EDTA, 125 mM NaCl). Samples were heated to
100 C° for 20 min and analyzed by SDS-PAGE and Western blotting. The
II spectrin bdp-1 antibody (35) was used at 1:10,000 in TBST (.01%
v/v Tween 20) for blotting.
I or
II spectrin or control peptides bound to
glutathione-agarose for 3 h at 4 C°, washed four times in above
buffer, and placed in sample buffer for immunoblotting with Ant-c-Src
(SRC2 Sc-18 Santa Cruz) at 1:1000 dilution in TBST.
II spectrin cDNA (GenBankTM accession
number U26396) by PCR DNA segments encoding six peptides. These
peptides correspond to the regions in human
II spectrin derived from
repeat units 9-12 and included i) its SH3 domain,
alternative insert 1, calpain cleavage site,
and its calmodulin-binding domain (peptide SACC, residues
913-1331); ii) its SH3 domain and alternative insert 1 (peptide SA,
residues 967-1077); iii) the SH3 domain with the tyrosine downstream
of insert 1 (peptide S+, residues 967-1077 (deleting
1053-1072), excluding alternative insert 1); iv) the SH3 domain alone
(peptide S, residues 967-1025); v) the calpain cleavage site and the
calmodulin-binding domain (peptide CC, residues 1172-1210); and vi)
the calmodulin-binding domain alone (peptide C, residues 1181-1210).
The oligonucleotides used were: for SACC, forward:
5'-cgcggatccgtgggcagcactgactatggc-3', reverse:
5'-ccggaattccttgcgctgatctgc-3'; for SA, S, and S+ forward: 5'-gtcggatccaaggagctggtcttggct-3', SA, reverse:
5'-gcgcggaattcatgatatagttctcccac-3'; for SH3 (S), reverse:
5'-ccggaattccggggtccaatttcttcac-3'; for S+, reverse:
5'-cggaattccttctcacccagttccagcagagaatgatactgattgtcaatctgctcctgccgcagtgc-3'; for CC, forward: 5'-ggatccagggatgaaactgattccaag-3'; for C, forward: 5'-gggatcccaacaggaagtgtatggcatg-3'; for CC and C, reverse:
5'-gggaattctctccttgatggaattaaaggtggc-3'. Amplification products
were subcloned into pGEX (Amersham Biosciences) and verified by
sequencing. Constructs were expressed as fusions with GST and purified
using glutathione-Sepharose as before (43). Molar equivalents of
recombinant peptides were incubated with 500 µM ATP in 75 mM MnCl2 in kinase reaction buffer (100 mM Tris-HCl, pH 7.2, 125 mM MgCl2,
25 mM MnCl2, 2 mM EGTA, 0.25 mM sodium orthovanadate, 2 mM DTT) with varying
amounts of c-Src kinase (Upstate Biotechnology) at 30 °C
for 3 h. Incorporation of ortho
-[32P]ATP was
measured by autoradiography after SDS-PAGE analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
II Spectrin in MDCK Cells Retards
Its Susceptibility to Calpain Digestion--
The phosphotyrosine
content of
II spectrin in confluent MDCK cells was examined by
immunoprecipitation and Western blotting with anti-phosphotyrosine
antibodies (Fig. 1A).
Immunoprecipitation was conducted under conditions that allow both
subunits of the strongly associated spectrin heterodimer (
II
II)
to be precipitated. Spectrin was abundant in the precipitates (Fig.
1A, left panel), and the immunoprecipitated
spectrin was reactive with antibodies to phosphotyrosine
(P-Y on Fig. 1A, center panel). The
level of anti-pTyr reactivity was increased by pretreatment of the
cells with vanadate, a tyrosine phosphatase inhibitor, but a
phosphotyrosine-modified spectrin was detectable even in untreated
cells. While we cannot exclude the presence of trace pTyr in
II
spectrin that is also present in these precipitates, the preponderance
of pTyr-modified spectrin, identified in multiple experiments
(n = 6), appears to be the
II subunit based on the
size of the pTyr immunoreactive band at
284 kDa. Tyrosine
phosphorylation of
II spectrin was also verified by
immunoprecipitation with anti-pTyr followed by Western blotting (Fig.
1A, right panel). Anti-pTyr precipitates revealed
the clear presence of
II spectrin, as well as a second immunoreactive band at
100 kDa. The identity of the latter band is
unknown and was not further characterized; it likely represents a
tyrosine-phosphorylated proteolytic product of
II spectrin.
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Fig. 1.
Tyrosine phosphorylation of spectrin in MDCK
cells reduces its calpain-mediated proteolysis. A,
confluent MDCK cells incubated for 30 min with or without pervanadate
were lysed, immunoprecipitated, and Western-blotted with either a
polyclonal antibody to II spectrin or with a monoclonal antibody to
phosphotyrosine (P-Y) (clone G10). Note that
immunoprecipitated spectrin is strongly reactive with anti-pTyr and
that
II spectrin is cleanly present in the anti-pTyr precipitates.
B, whole MDCK cell lysates evaluated with anti-bdp-1, a
polyclonal antibody specific for the µ-calpain cleavage product of
II spectrin, revealed in RIPA lysates an
32% reduction in the
level of spontaneous breakdown fragment at
150kDa after 30 min of
incubation with pervanadate.
II spectrin proteolysis
was low relative to intact spectrin, it was noted that with vanadate
treatment, the abundance of a spectrin breakdown fragment at
150 kDa
was consistently reduced (e.g. the heavily loaded lysate
lanes shown in Fig. 1, A and B). This 150-kDa
fragment is typically generated by calpain proteolysis of
II
spectrin at Tyr1176 (17). To verify relationship between
the level of this spontaneous spectrin break-down product and the
degree of tyrosine phosphorylation, the extent of
II spectrin
proteolysis was evaluated using an antibody specific for the
II
spectrin proteolytic fragment generated by calpain cleavage at
Tyr1176 (35). This antibody detected an
32% reduction
in the level of calpain-cleaved
II spectrin derived from cultured
MDCK cells after they had been exposed to vanadate for 30 min (Fig.
1B). To more thoroughly evaluate this putative in
vivo protection of
II spectrin from calpain cleavage,
additional experiments were conducted using maitotoxin to stimulate
calpain in quiescent MDCK cells (Fig. 2).
Maitotoxin is a marine toxin that opens L-type calcium channels and
specifically activates calpain but not caspase (39). It thus stimulates
the generation of the characteristic 150-kDa
II spectrin breakdown
product (
II-bdp1) (40). As with the extraction assays in Fig. 1,
incubation of MDCK cells with vanadate before maitotoxin exposure
protected
II spectrin from calpain cleavage (Fig. 2). This effect
directly correlated with the degree of tyrosine phosphorylation as
measured by pTyr antibody. The in vivo pTyr state of
II
spectrin is thus inversely proportional to its susceptibility to
calpain cleavage. Surprisingly, protection was not absolute since after
incubations exceeding 30 min, even in vanadate-treated cells, the level
of calpain-cleaved
II spectrin rose to control levels (Fig. 2). We
do not know the genesis of this biphasic effect but note that prolonged
exposure to maitotoxin is toxic to cells and, thus presumably at longer
times, creates a state no longer representative of the in
vivo environment.
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Fig. 2.
Vanadate protects II
spectrin from calpain-mediated breakdown in vivo.
Cultured MDCK cells were incubated up to 30 min with either buffer
alone or with 5 mM vanadate and then lysed directly into an
SDS-containing buffer. Precautions were taken to avoid post-lysis
proteolysis (see "Materials and Methods"). The extent of the
spectrin breakdown product (
II-bdp1) was then evaluated with an
antibody specific for this calpain cleavage product of spectrin. Note
that in the absence of maitotoxin, quiescent cells contain nearly
undetectable levels of
II-bdp1. Cleavage is stimulated by the
activation of calpain by maitotoxin and delayed by the tyrosine
phosphatase inhibitor vanadate, a treatment that increases the level of
tyrosine-phosphorylated
II spectrin. By 30 min of incubation
however, the protection afforded by vanadate is lost (for reasons that
remain uncertain).
II spectrin breakdown product (Fig. 2,
time zero). These results are in contrast to observations made
following alternative lysis procedures, such as preincubation in RIPA
buffer (Fig. 1A), and suggests that the presence of
II
spectrin bdp-1 in quiescent cultured cells as detected by ourselves
(Fig. 1A) and by others (14) probably represents an in
vitro (rather than in vivo) proteolytic event that must
occur rapidly following cell lysis.
II Spectrin in Vivo--
The
SH3 domain of
II spectrin flanks the site of calpain cleavage and
thus is an attractive candidate for the docking of kinases or
phosphatases involved in the control of
II spectrin cleavage by
calpain. An earlier report has documented by yeast two-hybrid assay
that isoform A of low-molecular-weight phosphotyrosine phosphatase
binds to this site (14). We have searched for direct binding partners
in MDCK lysates by GST pull-down assays with recombinant GST-spectrin
peptides containing only the SH3 domain of either
I or
II
spectrin (Fig. 3). Although the SH3
domains of
I and
II spectrin share 75% homology, only the SH3
domain of
II spectrin bound c-Src in the MDCK cell lysates.
Moreover, c-Src only bound when it was derived from vanadate-treated
cells, a condition that leads to its autophosphorylation. Although
these studies do not exclude the possibility of an unrecognized
intervening adapter protein, it seems likely that the tyrosine
phosphorylation of c-Src itself also determines its affinity for the
SH3 domain of
II spectrin. Similar requirements for the binding of
c-Src have also been noted in other settings (for review, see Ref. 41). The kinase c-Src thus makes an interesting complement to the
phosphotyrosine phosphatase that also binds to this SH3 domain in
II
spectrin (14).
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Fig. 3.
c-Src from vanadate-treated MDCK cell lysates
binds the SH3 domain of II spectrin.
Recombinant GST fusion peptides encompassing the SH3 domain of either
I or
II spectrin were incubated with MDCK cell lysates from
control and vanadate-treated cells. Western blots of the bound proteins
were then evaluated with anti-c-Src antibody. Note the presence of
c-Src in the MDCK cell lysates from both control and vanadate-treated
cells (arrow). Only the SH3 domain from
II spectrin, but
not the SH3 domain of
I spectrin, binds to the c-Src in the
lysates.
II Spectrin Peptides Are Phosphorylated in Vitro on
Tyr1073 and Tyr1176--
Observations in MDCK
cells indicated that
II spectrin could be tyrosine-phosphorylated
and that this event correlated with reduced
II spectrin proteolysis
by endogenous µ-calpain. While other mechanisms are certainly
possible, the simplest explanation of this linkage would be if
Tyr1176 is the target of phosphorylation, a modification
that would render it a less attractive substrate for µ-calpain (42).
In earlier work we have demonstrated that the critical
Tyr1176-Gly1177 bond targeted by µ-calpain
probably occurs in a highly exposed loop juxtaposed between helix C and
the calmodulin-binding domain within the 11th repeat unit of
II
spectrin, making it likely that subtle conformational features of this
region exert significant effects on its suitability as a calpain target
(43). This region of
II spectrin targeted by calpain is also
interesting in that adjacent to the calpain cleavage site and the
calmodulin-binding domain is a 20-residue sequence (insert 1) encoded
by alternative exon usage (44, 45). Upstream of this insert is the
spectrin SH3 domain, a motif common to many kinase and phosphatase
signal transduction cascades (46), and as shown above, a site that binds c-Src. We therefore focused on this region of
II spectrin. A
recombinant GST fusion peptide (SACC) encompassing
II spectrin repeats 9-12 was prepared (Fig.
4A). In silico
analysis by NetPhos 2.0 (47) predicted that of the eight tyrosine
residues in this peptide, only two were high-probability substrates for
tyrosine phosphorylation. One was indeed Tyr1176, the other
was Tyr1073 (Fig. 2B). Interestingly,
Tyr1073 is the first residue after insert 1, and its
phosphorylation potential is enhanced by sequences in insert 1 (Fig.
4B). When the purified recombinant GST-SACC peptide was
incubated in vitro with the tyrosine kinase c-Src and
-[32P]ATP, it became strongly labeled in a
dose-dependent manner (Fig. 4C). Parallel
studies using c-Src alone or a GST control peptide incorporated minimal
phosphate, confirming that the SACC peptide was a favorable substrate
for tyrosine phosphorylation.
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Fig. 4.
Tyrosine phosphorylation of SACC, a
spectrin peptide encompassing the calpain cleavage site.
A, schematic representing the recombinant fusion
protein SACC (SH3). The spectrin SH3 domain falls within the
repeat 9/10 segment. The alternative transcript (A) is a
20-residue sequence (insert 1) derived by alternative splicing. It is
variably and widely expressed in most tissues (45).
Asterisks mark the locations of the two sites of tyrosine
phosphorylation (see "Results"). The calpain cleavage site
(Cal) and the calmodulin-binding domain (CaM) are
novel sequences inserted into the putative third helix of spectrin
repeat 11 (43). B, in silico analysis of the
phosphorylation potential of SACC as analyzed by NetPhos 2.0 (47). This
graph is aligned with the schematic above it, showing the approximate
location of the eight tyrosine residues in SACC. Note that
Tyr1073 and Tyr1176 are predicted to be
favorable sites for phosphorylation and that the phosphorylation
potential of Tyr1073 is considerably reduced in the absence
of insert 1. C, increasing tyrosine phosphorylation of SACC
induced by increased amounts of recombinant c-Src. Inset,
SDS-PAGE analysis of SACC with c-Src and -[32P]ATP.
CB, Coomassie Blue-stained gel; AR,
autoradiogram.
II spectrin repeats 9 and
10, ± insert 1) were readily phosphorylated. Both of these peptides
contained Tyr1073. Conversely, peptide S was not
phosphorylated; it encompassed most of spectrin repeats 9 and 10 and
included five tyrosine residues and the SH3 domain but lacked
Tyr1073. Since Tyr1073 is the only tyrosine
present in peptides SA and S+ that is not present in peptide S, we
conclude that Tyr1073 is one target of spectrin tyrosine
phosphorylation in vitro. Surprisingly, despite the impact
of insert 1 on the calculated phosphorylation potential of
Tyr1073, we found that its presence or absence had little
effect on phosphorylation of Tyr1073 in these assays
(cf. Fig. 5B, lanes 2 and
3). Alternative phosphorylation conditions or kinases that
might reveal the predicted impact of insert 1 were not explored in the
present study.
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Fig. 5.
Identification of the sites of tyrosine
phosphorylation. A, schematic relationship of the
recombinant peptides. Note that only peptides with Tyr1073
or Tyr1176 are phosphorylated. B, Western blot
with anti-phosphotyrosine antibody demonstrating tyrosine
phosphorylation of the recombinant peptides. The position of the
auto-phosphorylated c-Src is marked (src). Lane
1, G-SACC (76 kDa); lane 2, G-SA (40 kDa); lane
3, G-S+ (35 kDa); lane 4, G-S (35 kDa); lane
5, GST alone (26 kDa); lane 6, G-CC (31 kDa);
lane 7, G-C (30 kDa). The graph depicts the relative extent
of phosphorylation compared with the level of c-Src phosphorylation.
Approximately equal protein loading of each recombinant protein was
verified by blotting with anti-GST antibody (data not shown).
II spectrin) revealed a second site of tyrosine phosphorylation.
These peptides differ only in the presence of the calpain cleavage
domain (Fig. 5A) with its single tyrosine, Tyr1176, at the calpain cleavage site. Peptide CC, but not
C, was readily tyrosine-phosphorylated (Fig. 3C), revealing
this critical tyrosine as a second favored site of phosphorylation.
II Spectrin
Peptide Susceptibility to Digestion by µ-Calpain--
After tyrosine
phosphorylation of the recombinant peptides SACC and CC, both
containing residue Tyr1176, their susceptibility to
in vitro proteolysis by increasing amounts of µ-calpain
was assayed by SDS-PAGE (Fig.
6A). These results were
quantified by densitometry of the stained gels (Fig. 6B). For both peptides, at every level of calpain, phosphorylation reduced
the fraction of peptide proteolysed. At maximum concentrations of
µ-calpain, tyrosine phosphorylation of SACC or CC afforded a 30-40%
reduction in the extent of cleavage by µ-calpain. Conversely, a
spectrin peptide representing the calmodulin-binding domain, but
lacking the calpain-sensitive Tyr1076 site, was not cleaved
by calpain (Fig. 6A, panel C). These results cannot be due to the presence of c-Src itself in the reaction mixture
since this kinase (without ATP) was included in all controls. It is
also worth noting that the significant reductions in calpain sensitivity observed here probably underestimate the degree of resistance conferred by tyrosine phosphorylation since it is unlikely that stoichiometric phosphorylation of Tyr1176 was
achieved.
View larger version (33K):
[in a new window]
Fig. 6.
Tyrosine phosphorylation of
Tyr1176 inhibits cleavage by
µ-calpain. A, SDS-PAGE analysis of
phosphorylated and non-phosphorylated II spectrin peptides subjected
to proteolysis with increasing amounts of µ-calpain. Peptides are
designated as in Fig. 5. PY marks the phosphorylated
peptide, and bdp represents its major µ-calpain-generated
cleavage product. Peptide C, which lacks the calpain sensitivity
region, is neither phosphorylated nor sensitive to calpain.
B, the relative extent of digestion was quantified by
densitometry. In every instance, phosphorylation of Tyr1076
retards susceptibility to calpain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
II spectrin i) is
tyrosine-phosphorylated in cultured MDCK cells and that the level of
this phosphorylation is increased by treatment with the tyrosine phosphatase inhibitor vanadate; ii) that the in vivo
susceptibility of endogenous
II spectrin to cleavage at
Tyr1176 by µ-calpain is inversely proportional to its
level of tyrosine phosphorylation; iii) that phosphorylated c-Src binds
to the SH3 domain of
II spectrin; iv) that the targets of in
vitro phosphorylation with c-Src kinase are residues
Tyr1073 and Tyr1176 in
II spectrin; and v)
that phosphorylation of Tyr1176 retards the susceptibility
of this site to cleavage by µ-calpain in vitro.
Collectively, these results establish the presence of at least two
sites of tyrosine phosphorylation in
II spectrin and identify a
novel regulatory mechanism acting through c-Src on the
µ-calpain-mediated processing pathway of spectrin. Given the growing
recognition of the importance of the cytoskeleton in determining
cellular function, both in organelles and at the plasma membrane, these
findings have implications for our understanding of membrane skeletal
control in several settings and indicate that spectrin may be a key
point of signal convergence between tyrosine kinase/phosphatase and
Ca2+-mediated signal cascades.
II spectrin might further protect
the integrity of the NMDA receptor complex by retarding proteolysis of
the spectrin membrane skeleton. While only a single study has explored
the impact of phosphatase inhibitors on glutamate receptor and spectrin stability (38) and found no protection of spectrin breakdown, our
present results suggest that this issue deserves a more complete exploration. Regardless, the results reported here establish a novel
post-translational modification in
II spectrin (tyrosine phosphorylation) and demonstrate a clear coupling between this modification and is susceptibility to calpain-mediated proteolysis, both in vivo and in vitro.
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ACKNOWLEDGEMENTS |
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We thank Mr. Paul Stabach for assistance with early aspects of this study and Dr. Susan Glantz for advice on the use of the cleavage-specific anti-spectrin antibodies and maitotoxin treatment of cell cultures.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (to J. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Depts. of Pathology and Molecular, Cellular, and Developmental Biology, Yale University, 310 Cedar St., New Haven, CT 06510. Tel.: 203-785-3624; Fax: 203-785-7037; E-mail: jon.morrow@yale.edu.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M210988200
1 S. P. Glantz, C. D. Cianci, K. K. Wang, J. S. Morrow, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: MDCK, Madin-Darby canine kidney; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TBST, (10 mM Tris-HCl (pH 8), 150 mM NaCl, and 0.05% Tween 20); GST, glutathione S-transferase; DTT, dithiothreitol; SH, Src homology; NMDA, N-methyl-D-aspartate receptor.
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