(Received for publication, June 22, 1995; and in revised form, July 24, 1995)
From the
Dynamic regulation of signal transduction by reversible palmitoylation-depalmitoylation cycles has been recently described. However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thioesterases involved. Here, we describe an assay for the palmitoyl acyltransferase (PAT) that palmitoylates ``myrGlyCys'' containing members of the Src family of protein tyrosine kinases (PTKs). Since N-myristoylation of Fyn PTK, a member of the Src family, has been shown to be a prerequisite for palmitoylation, a new single plasmid vector that allows overexpression of myristoylated Fyn substrate in Escherichia coli was developed. Purified myristoylated protein substrates were incubated with iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of bovine brain lysates. Transfer of radiolabel to the Fyn substrate was detected by SDS-polyacrylamide gel electrophoresis and autoradiography. This assay was used to partially purify and characterize PAT activity from bovine brain. Here, we demonstrate that PAT is a membrane-bound enzyme, which palmitoylates myristoylated Fyn substrates containing a cysteine residue in position three. The PAT activity attached palmitate to Fyn proteins via a thio-ester linkage and exhibited a fatty acyl CoA preference for long chain fatty acids. It is likely that palmitoylation of Fyn and other Src family members by PAT regulates PTK localization and signaling functions.
Modification of proteins by fatty acids greatly alters their structure, function, and subcellular localization and has recently been shown to be involved in several aspects of cellular signaling (see (1) and (2) for recent reviews). More than 200 proteins are known to be fatty acylated, including viral and cellular proteins(1, 2, 3) . Roles for protein fatty acylation range from anchoring proteins to membranes (3, 4) and stabilizing protein-protein interactions (5) to regulating enzymatic activities in mitochondria(6) . In general, mutations that prevent fatty acylation abolish or greatly alter the biological function of these proteins.
Protein fatty acylation can be divided into two
categories: myristoylation and palmitoylation. N-Myristoylation involves the cotranslational attachment of
the 14-carbon fatty acid myristate onto an N-terminal glycine residue
of a protein via an amide linkage. Due to the high stability of this
amide bond, myristoylation is irreversible, with some
exceptions(7) . The enzymology of N-myristoylation has
been well characterized ( (8) and references therein). A
methionyl aminopeptidase first removes the initiator methionine. N-Myristoyl transferase (NMT) ()then catalyzes the
transfer of myristate to the glycine residue in position two. This
glycine residue is an essential element of the substrate recognition
sequence, since its substitution by any other amino acid within a
protein prevents myristoylation.
Many proteins contain the
16-carbon fatty acid palmitate attached to specific cysteine residues.
In contrast to myristoylation, palmitoylation occurs
post-translationally and is readily reversible. The enzymology of
palmitoylation is not well characterized, and a palmitoyl
acyltransferase (PAT) has not yet been isolated. No apparent consensus
sequence has been found at the palmitoylation site, suggesting the
presence of more than one type of PAT. However, the N termini of 7 of 9
members of the Src family of PTKs and several of the subunits of
the heterotrimeric G proteins contain the sequence myrGlyCys, where
Cys-3 is palmitoylated(2, 3) . In most of these cases,
myristoylation has been shown to be a prerequisite for palmitoylation
to occur(9, 10) .
Many signal-transducing proteins
translocate reversibly between plasma membrane and
cytosol(1, 2) . Several have been shown to be
reversibly palmitoylated, such as subunits of G proteins and
nitric oxide synthase. These proteins undergo agonist-stimulated
palmitate turnover(11, 12) , suggesting that dynamic
palmitoylation of proteins can regulate signal transduction.
Little
is known about the enzymes that specifically remove or transfer
palmitate onto signal-transducing proteins. Recently, a palmitoyl
thioesterase, which deacylates Ras proteins and subunits of G
proteins in vitro, has been purified and the corresponding
cDNA cloned(13, 14) . Upon further analysis, this
palmitoyl thioesterase was shown to be a secreted protein. Ras proteins
and G
subunits, which are located on the cytoplasmic
layer of the plasma membrane, are therefore unlikely to be physiologic
substrates for a secreted palmitoyl thioesterase.
Progress toward
the identification of a PAT has been limited by lack of a rapid,
sensitive, and specific assay. Here, we report development of such an
assay for the PAT that palmitoylates the Src-related PTKs containing
the myrGlyCys mini-consensus sequence. Since prior myristoylation of
these proteins has been shown to be a prerequisite for palmitoylation,
a source of myristoylated protein to be used as substrate in the assay
was required. An Escherichia coli overexpression vector for
protein myristoylation was used to produce large amounts of
myristoylated truncated Fyn PTK proteins. To circumvent long exposure
times required to detect incorporation of tritiated palmitate into
proteins, a [I]iodopalmitoyl CoA analog was
utilized(15, 16) . Analysis of the products of the PAT
assay by SDS-PAGE allowed visualization of incorporated iodopalmitate
in as little as 30 min using phosphorimager technology. This assay was
used to partially purify and characterize a PAT activity from bovine
brain that palmitoylates myristoylated proteins.
Figure 1:
Design of
an artificial operon for tandem expression of truncated Fyn
protein-tyrosine kinase constructs and human N-myristoyl
transferase in E. coli. A, The pETFyn432hNMT plasmid
is shown with the sequence of the intercistronic region containing the
Shine-Dalgarno (SD) sequence. B, N-terminal sequences
of WT and mutant Fyn engineered in the FynSH432His protein.
The substituted amino acids are underlined.
120 g of frozen suspension
(equivalent to 80 g bovine brain) was crushed with a mortar
and pestle and thawed in 500 ml of cold H buffer. The brain tissue was
homogenized twice for 30 s in a Waring blender with a 5-min rest
interval. The homogenate (H) was centrifuged at 10,000 g for 15 min to remove the cellular debris and the
mitochondria/nuclei containing fraction (P10). The supernatant was
carefully transferred to a fresh container, and the pellet was
reextracted with 500 ml of H buffer as described above.
The second
supernatant was pooled with the first, and the resulting suspension
(1000 ml) was centrifuged at 100,000 g for 45 min. The
supernatant (S100) was decanted, and the pelleted membrane fraction
(P100) was resuspended in 100 ml of buffer H with five up and down
cycles on a motor-driven Potter-Elvehjem homogenizer. The suspension
was adjusted to 100 mM Na
CO
at pH 11.0
and stirred for 30 min. Membranes were pelleted by centrifugation at
100,000
g for 45 min. The carbonate-washed membranes
were resuspended in 50 ml of buffer H using the motor-driven
homogenizer as described above, and the pH of suspension was adjusted
to 8.5.
The membrane suspension was adjusted to 25% (v/v) glycerol, and membranes were solubilized with 1% (w/v) Triton X-100 at a detergent to protein ratio (w/w) of 3. The suspension was stirred for 30 min and then centrifuged as above for 45 min. The supernatant was transferred to new tubes and kept frozen at -80 °C until needed. Subcellular fractions were assayed for activity as described below.
To study substrate specificity, 2.5 µg of partially
purified PAT from the Q-Sepharose pool was assayed as above in the
presence of 1.0 µg of the purified FynSH432His mutants.
Alternatively, PAT activity was assayed with the WT FynSH432His
in the presence of myristoylated dodecapeptides (100
µM) corresponding to the N termini of Src-related protein
tyrosine kinases(19) .
To study temperature sensitivity of PAT activity, 2.5 µg of partially purified PAT (Q pool) were preincubated for 5 min at 25, 37, 45, 55, 75, and 100 °C, chilled for 5 min on ice, and assayed for activity. The fatty acyl CoA specificity was investigated by assaying PAT activity in the presence of fatty acyl CoAs (10 µM) of increasing chain length.
To ensure that the palmitoylation assay was specific for the
N-terminal region of the FynSH432His protein, a series of
mutations was engineered at the Fyn N terminus. As shown in Fig. 1B, cysteine residues were substituted for serine
residues individually (C3S and C6S) and together (C3,6S). In addition,
the myristoylation signal was abolished by substituting the essential
glycine residue at position two with an alanine residue (G2A). A SrcFyn
chimera containing the first 10 amino acids of Src replacing the
corresponding Fyn N-terminal region was also engineered as a negative
control, since the Src N terminus is known not to be
palmitoylated(9) . All the mutations and the engineered stretch
of DNA were fully sequenced, and no mutations other than those
engineered were found in the clones utilized in this study.
Figure 2:
Overexpression, purification, and
myristoylation of FynSH432His proteins in E. coli. A, Coomassie-stained SDS-polyacrylamide gel of aliquots from a
typical purification of the truncated Fyn proteins by nickel-chelating
chromatography. The arrow indicates the position of the
FynSH432His
proteins. Lane1, molecular
mass (MW) markers (top to bottom, 106, 80,
49.5, 32.5, 27.5, and 18.5 kDa); lane2, solubilized
lysates from E. coli overexpressing the WT Fyn construct; lane3, nickel-chelating column flow-through (FT); lanes4-9, 2.0 µg of
purified WT, C3S, C6S, C3,6S, G2A, and SrcFyn FynSH432His
proteins. B, autoradiogram of
[
H]myristate-labeled E. coli overexpressing FynSH432His
proteins. Equivalent
amounts of SDS-solubilized E. coli protein lysates were
separated by SDS-PAGE on a 12.5% acrylamide gel. Lanes shown are WT,
C3S, C6S, C3,6S, G2A, and SrcFyn FynSH432His
proteins and
vector without insert control(-). Exposure time, 8
h.
To scale up the purification of PAT and to
determine its intracellular localization, a bovine brain homogenate was
fractionated into membrane-bound and soluble compartments. Membrane
fractions contained the most PAT activity, while the cytosolic fraction
was devoid of detectable activity (Fig. 3). The 100,000 g pellet (P100) was washed with carbonate (pH 11) to remove
peripheral membrane proteins. PAT activity was clearly detected in the
carbonate-washed membranes and could readily be extracted with
detergent (Fig. 3). Solubilization of PAT from the low speed,
P10 membrane fraction was inefficient.
Figure 3:
Subcellular fractionation of bovine brain
PAT and partial purification of the solubilized PAT. Different
subcellular fractions (2.5 µg) were prepared and assayed for PAT
activity as described under ``Materials and Methods.'' The
position of FynSH432His substrates is indicated by the arrow. PAT assays were carried out with WT (odd-numbered
lanes) and C3,6S (even-numbered lanes) FynSH432His
proteins as substrates. Products of the reactions were separated
by SDS-PAGE, and [
I]iodopalmitate analog
incorporation into FynSH432His
proteins was visualized by
autoradiography. H, homogenate; P10, 10,000
g pellet; P100, 100,000
g pellet; S100, 100,000
g supernatant; C.W.P100, carbonate-washed membranes; Sol. PAT, 1.0%
Triton X-100-solubilized carbonate-washed P100 membranes; and Q
pool, Q-Sepharose pool (see Fig. 4for chromatographic
details). Lanes 15 and 16, substrate alone controls (no enzyme added).
Exposure time was 36 h.
Figure 4: Chromatographic elution profile of PAT activity on Q-Sepharose Fast Flow, pH 8.5. PAT activity was assayed in the different fractions and quantified using phosphorimager analysis.
Solubilized PAT was
fractionated on a Q-Sepharose Fast Flow column at pH 8.5 and eluted
between 175 and 300 mM NaCl (Fig. 4). This PAT pool was
used in all subsequent assays. The PAT activity was very labile,
thereby complicating its purification. Indeed, enzymatic activity could
not be measured after storage for more than 10 days at 4 °C, and
the enzyme did not tolerate multiple freeze/thaw cycles. ()
Figure 5:
Substrate specificity of the PAT. A, 2.5 µg of partially purified PAT (Q-Sepharose pool)
were assayed with the various FynSH432His protein
substrates as described under ``Materials and Methods.''
[
I]Iodopalmitate analog incorporation into
FynSH432His
proteins was visualized by autoradiography. 1.0
µg of WT, C3S, C6S, C3,6S, G2A, and SrcFyn-truncated Fyn proteins
were used as substrates in the palmitoylation reaction. WT substrate
alone and enzyme alone were used as negative controls (lasttwolanes). Exposure time, 36 h. B, PAT
activity was measured in the absence and presence of various
dodecapeptides (100 µM) corresponding to the N termini of
protein-tyrosine kinases of the Src family(19) . WT substrate
alone and enzyme alone were used as negative controls (lasttwolanes). C, the PAT preparation was
incubated for 5 min at the indicated temperature, assayed as described
under ``Materials and Methods'' with WT FynSH432His
substrate, and analyzed by SDS-PAGE and autoradiography. Exposure
time, 36 h.
The G2A Fyn mutant substrate, which is not myristoylated, was not palmitoylated in vitro, despite the presence of cysteines at positions three and six. This result suggests that myristoylation is a prerequisite for palmitoylation. When PAT was assayed in the presence of myristoylated dodecapeptides corresponding to the N termini of Src-related PTKs (Fig. 5B), only the peptides containing the N-terminal sequence myrGlyCys inhibited the reaction. Indeed, the PAT activity was inhibited greater than 95% by 100 µM myrFyn and myrLck, while myrLyn and myrYes peptides inhibited the reaction by 84 and 69%, respectively. Interestingly, peptides containing two cysteines (myrFyn and myrLck) were better inhibitors. No inhibition by the myrSrc peptide (which has no cysteine residue in its sequence) nor by the non-myristoylated Yes peptide was observed. These observations imply that proteins containing the myrGlyCys motif are substrates for palmitoylation by PAT.
Other
investigators have reported that non-enzymatic palmitoylation of
proteins and peptides can occur at physiological
pH(29, 30) . However, under our assay conditions, the
protein substrate (WT) alone and enzyme alone controls did not reveal
any significant incorporation of the labeled palmitate analog in the
region corresponding to FynSH432His substrate ( Fig. 3and Fig. 5A and 5B). In addition,
PAT activity was heat labile, as preincubation of the PAT preparation
at 45 °C for 5 min completely prevented palmitoylation (Fig. 5C). These results indicate that PAT activity is
enzymatic.
Figure 6:
Fatty acyl CoA specificity of the PAT. 2.5
µg of partially purified PAT (Q-Sepharose pool) was assayed as
described under ``Materials and Methods,'' and
[I]iodopalmitate analog incorporation into WT
FynSH432His
protein substrate was visualized by
autoradiography. A, assays performed in the absence or
presence of non-radioactive fatty acyl CoAs of increasing chain length. B) PAT was assayed with the WT substrate (odd-numbered
lanes) or with the C3,6S mutant substrate (even-numbered
lanes), and the products of the reaction were analyzed by
SDS-PAGE. In situ gel hydrolysis of bound radioactivity was
performed as described under ``Materials and Methods'' by
incubating the gels with neutral hydroxylamine (lanes3, 4, 7, and 8) or with Tris
buffer as control (lanes1, 2, 5,
and 6). Lanes1-4,
Coomassie-R250-stained gels; lanes5-8,
corresponding autoradiogram. The arrow shows the position of
the FynSH432His
substrates. Exposure time, 36 h. C, radioactivity released from the FynSH432His
substrate after alkali hydrolysis was analyzed by reversed phase
TLC. Lane1, FynSH432His
hydrolysate; lane2,
I-labeled IC16 standard; lane3, hydrolysate and standard mixed together.
Exposure time, 8 days. ori, sample
origin.
In a parallel experiment, the
palmitoylated substrate was excised from the gel and hydrolyzed with
alkali. The hydrolysate was neutralized, chloroform extracted, and
analyzed by thin layer chromatography. As shown in Fig. 6C, the mobility of the sample derived from the
hydrolysate was identical to that of the
[I]iodopalmitate analog standard, thereby
identifying the fatty acid bound to the FynSH432His
substrate as the iodopalmitate analog. In lane3 of the same chromatogram, the hydrolysate sample and iodopalmitate
standard were spotted together, yielding a single spot of increased
intensity after development of the TLC. This latter result further
confirmed the identity of the iodopalmitate analog as the transferred
fatty acid.
In this manuscript, we report a rapid and sensitive assay for
the PAT that palmitoylates myrGlyCys containing proteins of Src-related
PTKs and its use in the purification and characterization of a PAT
activity from bovine brain. The assay employs a high energy
radiolabeled [I]iodopalmitoyl CoA analog and a
variety of highly purified myristoylated FynSH432His
protein substrates. Detection of PAT activity was accomplished
with less than 1 h of exposure of dried SDS-PAGE gels on a
phosphorimager screen using 2.5 µg of protein or less. This
represents a significant improvement over previous palmitoylation
assays, which required a minimum of 48 h of exposure using tritiated
palmitate and 50 µg of PAT extract to visualize palmitate
incorporation into viral glycoproteins(31) .
A series of
myristoylated Fyn substrates was overexpressed in E. coli with
a single plasmid myristoylation system and used to delineate the
substrate specificity of PAT. All FynSH432His substrates
were shown to be expressed and myristoylated to equivalent levels
(except G2A, which lacks the myristoylation signal) (Fig. 2). N-Myristoylation was shown to be essential for palmitoylation in vitro and is likely to be part of the recognition signal
for PAT within the Fyn sequence. Two lines of evidence support this
conclusion: 1) the G2A mutant FynSH432His
substrate, which
is not myristoylated, is also not palmitoylated (Fig. 5A) and 2) only the myristoylated form of the Yes
dodecapeptide was an inhibitor of the PAT reaction (Fig. 5B). These results confirm previous observations
made in vivo, suggesting that myristoylation of the MetGlyCys
sequence of Src-related PTKs and G
subunits was a
prerequisite for palmitoylation(9, 10) . It is not
known whether other lipid moieties can substitute for myristate.
Recently, Degtyarev et al.(32) reported that
membrane association, rather than myristoylation, was required for
palmitoylation of Gin vivo. Likewise, it is
important to note that G
, which is not myristoylated,
is still palmitoylated at Cys-3(33, 34) . Although our in vitro assays are performed in cell-free lysates, in the
absence of membranes, the assay buffer does contain 0.3% Triton X-100,
a detergent concentration above the critical micelle concentration. The
presence of a myristate moiety could enhance the ability of the Fyn
substrate to productively interact with PAT in a detergent micelle.
Alternatively, Fyn and G
proteins may be palmitoylated
by different PATs in vivo with different substrate
specificities.
Using the cell-free assay and the various
FynSH432His substrates, we delineated the substrate
requirements of the partially purified PAT (Q-Sepharose pool). The
presence of Cys-3 was required for palmitoylation of Fyn in
vitro, since the C3S substitution within the FynSH432His
substrate completely abolished palmitoylation (Fig. 5).
Likewise, the SrcFyn chimera, which lacks Cys-3, was not palmitoylated,
and myrSrc peptide failed to inhibit the PAT activity. It is not known
whether sequences containing cysteine at positions 4 or 5 can serve as
PAT substrates.
Mutation of Cys-6 within Fyn did not inhibit
palmitoylation in vitro. In contrast, C6S mutants of
p59 expressed in vivo exhibit reduced levels of
palmitoylation(9) . It is possible that additional PATs are
responsible for palmitoylating Cys-6 in vivo or that the
absence of Cys-6 in the C6S mutant enhances palmitoylation of Cys-3 in vitro. Alternatively, differences observed in
palmitoylation of C6S mutants may reflect the use of monkey cells
(COS-1) versus bovine brain.
The PAT activity also
transferred iodopalmitate onto two other chimeric substrates:
YesFynSH432His and a
G
FynSH432His
. (
)Since no
homology could be found among the N termini (10-amino acid sequence) of
Fyn, Yes, and G
other than that encoding for the
myristoylation signal and the presence of the cysteine in position
three, the myrGlyCys sequence is likely to be necessary and sufficient
to direct palmitoylation.
The PAT activity exhibited selectivity
for long chain fatty acyl CoAs, as demonstrated by the inhibitory
properties of the various acyl CoAs in the assay (Fig. 6). Only
fatty acyl CoAs containing 14 carbons or more were significant
inhibitors of the PAT, palmitoyl CoA being the best inhibitor and the
likely natural substrate of the enzyme. Whether PAT activity can
transfer shorter or longer fatty acids onto proteins in vivo remains to be tested. The transfer of
[I]iodopalmitate to FynSH432His
occurred via the formation of an alkali and
hydroxylamine-sensitive linkage, indicative of a thioester bond. Taken
together, these data support the conclusion that PAT catalyzes
attachment of palmitate to Cys-3 in the Src family members.
The PAT
activity was shown to be membrane bound upon subcellular fractionation
of bovine brain (Fig. 3), consistent with the membrane
localization of Src-related PTKs and that of G proteins. Other PAT activities have also been found in membrane
fractions, e.g. the PAT that palmitoylates viral glycoproteins
and H-Ras(31, 35) . Whether these PAT activities
correspond to identical or different enzymatic activities is not known
at the present. The identity of the particular membrane subtype
containing the PAT activity is also not known. PAT and its substrates
could potentially interact at specific membrane sites. Upon palmitate
transfer, the palmitoylated protein would remain stably anchored in the
membrane. Identification of membranes enriched for PAT might shed light
on how these myrGlyCys-containing proteins are targeted to specific
intracellular membranes.
PAT activity could be efficiently solubilized from cellular membranes and fractionated on a Q-Sepharose Fast Flow column ( Fig. 3and Fig. 4). However, recovery of the PAT activity from the Q-Sepharose column was low (about 25%) and the increase in specific activity modest (typically 3-4-fold at best). Similar results were obtained with various chromatographic media. These observations, combined with the fact that PAT activity is very labile, have complicated further purification.
In conclusion, dynamic palmitoylation of proteins is a novel and exciting addition to the repertoire of cellular control mechanisms. To further understand this new mechanism, purification, and characterization of the PAT and palmitoyl thioesterase enzymes is imperative. To attain these goals, we designed and utilized a sensitive assay to partially purify and characterize a PAT activity that palmitoylates members of the Src family of PTKs. Once this PAT activity is purified to homogeneity, its encoding cDNA will be cloned, thereby allowing characterization at the molecular level.