(Received for publication, November 8, 1995)
From the
Caveolae are flask-shaped plasma membrane specializations that
are thought to exist in most cell types. A 22-kDa protein, caveolin, is
an integral membrane component of caveolae membranes in vivo.
Previous studies have demonstrated that caveolin is phosphorylated on
tyrosine by oncogenic viral Src (v-Src) and that caveolin is physically
associated as a hetero-oligomeric complex with normal cellular Src
(c-Src) and other Src family tyrosine kinases. Caveolin contains eight
conserved tyrosine residues that may serve as potential substrates for
Src. Here, we have begun to study the phosphorylation of caveolin by
Src family tyrosine kinases both in vitro and in
vivo. Using purified recombinant components, we first
reconstituted the phosphorylation of caveolin by Src kinase in
vitro. Microsequencing of Src-phosphorylated caveolin revealed
that phosphorylation occurs within the extreme N-terminal region of
full-length caveolin between residues 6 and 26. This region contains
three tyrosine residues at positions 6, 14, and 25. Deletion
mutagenesis demonstrates that caveolin residues 1-21 are
sufficient to support this phosphorylation event, implicating tyrosine
6 and/or 14. In vitro phosphorylation of caveolin-derived
synthetic peptides and site-directed mutagenesis directly show that
tyrosine 14 is the principal substrate for Src kinase. In support of
these observations, tyrosine 14 is the only tyrosine residue within
caveolin that bears any resemblance to the known recognition motifs for
Src family tyrosine kinases. In order to confirm or refute the
relevance of these in vitro studies, we next analyzed the
tyrosine phosphorylation of endogenous caveolin in v-Src transformed
NIH 3T3 cells. In vivo, two isoforms of caveolin are known to
exist: -caveolin contains residues 1-178 and
-caveolin
contains residues 32-178. Only
-caveolin underwent tyrosine
phosphorylation in v-Src transformed NIH 3T3 cells, although
-caveolin is well expressed in these cells. As
-caveolin
lacks residues 1-31 (and therefore tyrosine 14), these in
vivo studies directly demonstrate the validity of our in vitro studies. Because
- and
-caveolin are known to assume a
distinct but overlapping subcellular distribution within a single cell,
v-Src phosphorylation of
-caveolin may only affect a subpopulation
of caveolae that contain
-caveolin.
Caveolae are specialized domains of the plasma membrane(1, 2) . They are most numerous in endothelial cells, fibroblasts, adipocytes, and smooth muscle cells, although they appear to exist in most cells (3) . Their exact function remains unknown, although they have recently been implicated in certain transmembrane signaling events, including G protein-coupled signaling(1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo(14) . Several independent lines of evidence suggest that caveolin may act as a scaffolding protein within caveolae membranes. In support of this assertion: (i) both the N-terminal and C-terminal domains of caveolin face the cytoplasm(15) ; (ii) caveolin exists within caveolae membranes as a high molecular mass homo-oligomer(16, 17) ; (iii) these caveolin homo-oligomers have the capacity to self-associate in vitro to form larger structures that resemble caveolae(16) ; (iv) caveolin co-purifies with cytoplasmic signaling molecules including trimeric G proteins, Src family tyrosine kinases, and Ras-related GTPases(4, 5, 6) ; (v) recombinant caveolin interacts directly with hetero-trimeric G proteins(10) ; and (vi) caveolin binding can functionally suppress the GTPase activity and GTP binding of purified trimeric G proteins, holding the G protein in the inactive conformation(10) . Thus, caveolin may serve as an oligomeric docking site for organizing and concentrating inactive signaling molecules within caveolae membranes (16) .
Modification and/or inactivation of caveolin may be a common feature of the transformed phenotype(11) . Historically, caveolin was first identified as a major v-Src substrate in Rous sarcoma virus-transformed cells(18) . Caveolin is one of only a few known transformation-dependent v-Src substrates; both cell transformation and tyrosine phosphorylation of caveolin by v-Src are critically dependent on membrane attachment of Src via N-terminal myristoylation(19) . Based on these observations, Glenney and co-workers have proposed that caveolin may represent a critical target during cellular transformation. In direct support of this notion, caveolin expression and caveolae are dramatically reduced or absent in cells transformed by activated oncogenes other than v-Src (v-Abl, Bcr-Abl, and activated Ras)(11) .
The functional consequences of the phosphorylation of caveolin on tyrosine are not yet known. At steady state, caveolin is not phosphorylated on tyrosine(12, 20) . This is in contrast to v-Src-transformed cells where caveolin is constitutively phosphorylated on tyrosine(19) . However, caveolin tyrosine phosphorylation also occurs in normal cells but in a tightly regulated fashion(12) . If 3T3-L1 adipocytes are serum-starved and rapidly stimulated with insulin, caveolin transiently undergoes phosphorylation on tyrosine in a time- and dose-dependent manner(12) . It has been suggested that insulin-stimulated tyrosine phosphorylation of caveolin occurs indirectly via an endogenous Src family tyrosine kinase, rather than directly via the insulin receptor. In support of this idea, caveolin can be purified as part of a hetero-oligomeric complex that contains c-Src and other Src family tyrosine kinases(4, 5) .
Here, we have begun to study the tyrosine phosphorylation of caveolin by Src family tyrosine kinases both in vitro and in vivo. We first reconstituted the tyrosine phosphorylation of caveolin by c-Src in vitro. Our results indicate that tyrosine 14 of caveolin is the principal site that is recognized by the Src kinase in vitro. In addition, we also demonstrated the in vivo relevance of these in vitro studies using v-Src-transformed NIH 3T3 cells that endogenously express caveolin.
In order to understand the potential role of caveolin in
v-Src transformation, we have begun to map the site or sites of
caveolin tyrosine phosphorylation by Src. Caveolin contains eight
tyrosine residues that are conserved across species and could serve as
potential sites for tyrosine phosphorylation (Tyr,
Tyr
, Tyr
, Tyr
,
Tyr
, Tyr
, Tyr
, and
Tyr
; see Tang et al. for an
alignment(24) ). Note that tyrosine 42 is found only in canine
caveolin.
Figure 1:
In vitro phosphorylation of recombinant full-length caveolin by Src kinase.
Purified full-length caveolin (GST-FL-Cav) expressed as a GST
fusion protein or GST alone were subjected to in vitro phosphorylation with purified c-Src and
[-
P]ATP in kinase reaction buffer. After
SDS-PAGE, phosphorylated proteins were visualized by autoradiography.
Note that each reaction contained equivalent amounts of GST and
purified full-length caveolin, although only caveolin undergoes
phosphorylation. Phosphorylation of purified full-length caveolin was
specifically dependent on addition of both c-Src and
[
-
P]ATP. Omission of either c-Src or
[
-
P]ATP prevented phosphorylation (not
shown).
To identify a region of caveolin that is phosphorylated, we digested Src-phosphorylated caveolin with the endoproteinase Lys-C and separated the peptides by HPLC. A total of thirteen peptides are expected from digestion of caveolin with Lys-C; only four of these peptides contain tyrosine residues (Table 1). Eighty column fractions were collected, and a single column fraction containing a radiolabeled peptide was identified (Fig. 2, A and B). Microsequencing of this peptide revealed that caveolin is phosphorylated at its extreme N terminus (tyrosine at amino acid position 6, 14, or 25) (Fig. 2C).
Figure 2: Microsequence analysis of Src-phosphorylated caveolin. Caveolin was phosphorylated by c-Src as in Fig. 1and subjected to microsequence analysis as we have described previously for other proteins(5, 21) . Briefly, after SDS-PAGE and transfer to nitrocellulose, the caveolin band was excised and digested with Lys-C. A, after digestion, peptides were separated by HPLC and 80 column fractions were collected. B, an aliquot of each column fraction was then spotted onto polyvinylidene difluoride membranes and subjected to autoradiography. Only fraction 34 contained a radiolabeled peptide. C, microsequencing of this peptide revealed that it corresponds to caveolin residues 6-26. This indicates that tyrosine residues 6, 14, and/or 25 are the most likely sites for Src phosphorylation.
Because our microsequencing results suggest that the extreme N terminus of caveolin is the target for Src phosphorylation, we evaluated whether various portions of the N-terminal caveolin domain expressed as GST fusion proteins were capable of supporting Src-mediated phosphorylation. Fig. 3shows that caveolin residues 1-21 are sufficient to undergo Src-mediated phosphorylation. This suggests that tyrosine at either position 6 or position 14 is the primary site of Src phosphorylation. These observations are also consistent with our results from microsequence analysis.
Figure 3: Caveolin residues 1-21 are sufficient for phosphorylation of caveolin by Src. Recombinant fusion proteins encoding various portions of the N-terminal cytoplasmic domain of caveolin (A, residues 1-101; B, residues 1-21, 1-41, 1-81, and 61-101) or GST alone were subjected to in vitro phosphorylation by purified c-Src kinase. Each reaction contained equivalent amounts of GST and GST-caveolin fusion proteins. Src-phosphorylated proteins were visualized after SDS-PAGE by autoradiography. Note that caveolin residues 1-21 are sufficient to support Src phosphorylation, whereas residues 61-101 are not phosphorylated.
Figure 4:
Tyrosine 14 is the principal site of
phosphorylation by Src kinase in vitro. A, tyrosine
phosphorylation of caveolin-derived peptides by Src kinase.
Caveolin-derived peptides 1-8 (detailed in Table 2) were
directly synthesized as discrete spots on a membranous support. These
immobilized peptides were then incubated with purified Src kinase in
the presence of 1 mM ATP. Phosphotyrosine residues were
visualized by immunoblotting with the mAb 4G10. Only peptide 2 (the
Tyr-containing peptide) underwent tyrosine phosphorylation
under these conditions. B, in vitro phosphorylation
of the Y14F mutant by Src kinase. Recombinant GST-fusion proteins
containing residues 1-41 of the N-terminal domain of caveolin
were subjected to in vitro phosphorylation by purified c-Src
kinase. WT, wild type; Y14F, tyrosine 14 to
phenylalanine mutant. Each reaction contained equivalent amounts of
wild type and the Y14F mutant. Src-phosphorylated proteins were
visualized after SDS-PAGE by autoradiography. Note that the Y14F mutant
does not undergo phosphorylation, although it contains tyrosine
residues 6 and 25.
To confirm the relative importance of tyrosine 14 as a substrate for Src phosphorylation, we used a second independent approach. We mutated tyrosine 14 of GST-caveolin to phenylalanine by site-directed mutagenesis (Y14F). In accordance with the above peptide experiments, in vitro kinase assays revealed that the Y14F mutant failed to undergo phosphorylation by Src (Fig. 4B). This suggests that tyrosine 14 is the primary site of Src phosphorylation in vitro. These results are also consistent with our results independently obtained through microsequencing and deletion mutagenesis.
Because
our in vitro data suggest that Src phosphorylates caveolin at
its extreme N terminus on tyrosine 14, our results predict that in
vivo only -caveolin should be phosphorylated by Src because
-caveolin lacks tyrosine 14 because it does not contain caveolin
residues 1-31. To test this prediction, we immunoprecipitated
caveolin from v-Src-transformed NIH 3T3 cells and blotted these
immunoprecipitates with an antibody (4G10) that recognizes
phosphotyrosine residues. Fig. 5shows that although both
-
and
-caveolin isoforms are present in these immunoprecipitates,
-caveolin is selectively phosphorylated on tyrosine. These results
directly demonstrate the in vivo relevance and the validity of
our in vitro studies.
Figure 5:
Only the -isoform of caveolin is
phosphorylated on tyrosine in v-Src transformed NIH 3T3 cells in
vivo.A, schematic diagram showing the positions of the
two start sites within the caveolin coding sequence and the resulting
caveolin isoforms that differ in their extreme N-terminal protein
sequence. Note that only
-caveolin contains tyrosine 14.
-Caveolin specifically lacks tyrosine 14 because it does not
contain caveolin residues 1-31. B, v-Src-transformed NIH
3T3 cells were solubilized in a buffer containing octyl-glucoside and
tyrosine phosphatase inhibitors and subjected to immunoprecipitation
with a rabbit polyclonal antibody that recognizes both
- and
-caveolin isoforms. After SDS-PAGE and transfer to nitrocellulose,
caveolin immunoprecipitates were probed with a monoclonal antibody that
recognizes phosphotyrosine residues (4G10) or another monoclonal
antibody(2297) that recognizes both
- and
-caveolin
isoforms.
The oncogene v-Src arose by viral transduction of the normal cellular gene c-Src(25, 26) . Thus, it is thought that viral tyrosine kinases largely induce transformation by intercepting cell regulatory mechanisms that are normally under the control of tyrosine phosphorylation. In support of this notion, v-Src and c-Src appear to differ primarily in enzymatic activity but not in their substrate specificity(27, 28) . For example, both c-Src and v-Src phosphorylate Ras GAP at the same major and minor sites both in vitro and in vivo(29, 30) . This difference in enzymatic activity can be attributed to the loss of tyrosine residue 527 within v-Src. Phosphorylation at this C-terminal site within c-Src by CSK kinase normally inactivates c-Src(31, 32) .
Few transformation-dependent v-Src substrates are known. Caveolin is one of these transformation-dependent substrates. Tyrosine phosphorylation of caveolin by v-Src requires the membrane attachment of v-Src via myristoylation(19) . Similarly, membrane attachment of v-Src is required for its transforming activity. Thus, Glenney (19) has suggested that caveolin may represent a critical target for v-Src. Also, caveolin co-purifies as a hetero-oligomeric complex with c-Src and other Src family tyrosine kinases(4, 5) . This is consistent with the general idea that v-Src phosphorylates the normal targets of c-Src or related Src family tyrosine kinases but in an unregulated fashion.
Here, we have reconstituted the tyrosine phosphorylation of caveolin in vitro using purified recombinant components: c-Src and GST-caveolin fusion proteins. By using a combination of microsequencing, deletion mutagenesis, synthetic peptide substrates, and site-directed mutagensis, this in vitro approach has allowed us to identify tyrosine 14 within caveolin as the principal site of phosphorylation by Src. The specific phosphorylation of a given tyrosine residue by Src family kinases is dependent on the surrounding amino acid sequences(28) . In this regard, it is interesting to note that tyrosine 14 is the only tyrosine-containing site within caveolin that bears any homology to the known consensus sites for v-Src and c-Abl tyrosine kinases (Fig. 6).
Figure 6: Comparison of caveolin tyrosine 14 and its surrounding sequence with the preferred recognition sequences of tyrosine kinases. The amino acid sequence of canine caveolin (residues 9-18) is compared with the preferred recognition sequences of c-Abl and v-Src(28) . Identical residues and conservative substitutions are boxed. An asterisk indicates that a given caveolin residue is identical in all caveolin molecules across species (from chick to man). A dash indicates that a given caveolin residue is not identical across species but undergoes a conservative substitution. Note that the substrate preferences for v-Src and c-Src are identical and are detailed in Songyang et al.(28) .
Tyrosine 14 is present
at the extreme N terminus of full-length caveolin. However, two
isoforms of caveolin exist that differ in their extreme N
terminus(21) . These two isoforms derive from a single gene
through alternate initiation during translation; methionine 32 serves
as an internal start site to generate -caveolin(21) .
Therefore,
-caveolin contains the complete caveolin sequence,
whereas
-caveolin lacks residues 1-31 and therefore tyrosine
14. Because
-caveolin specifically lacks tyrosine 14, it should
not undergo phosphorylation by Src. In direct support of this
prediction, we observed that
-caveolin is selectively
phosphorylated on tyrosine in v-Src-transformed NIH 3T3 cells, although
these cells express both
- and
-caveolin. Thus, it appears
that
-caveolin is actually the target of v-Src rather than
-caveolin. This is the first time this distinction has been
delineated. This may be important because
- and
-caveolin
assume a distinct but overlapping subcellular distribution within a
single cell, implying the existence of subpopulations of
caveolae(21) . In addition,
-caveolin is preferentially
phosphorylated on tyrosine in response to insulin stimulation in
vivo, although this was not noted by the authors because the
structural differences between
- and
-caveolin were not known
at the time(12) . This insulin-stimulated tyrosine
phosphorylation of caveolin is thought to occur via an endogenous
member of the Src family of tyrosine kinases (12) rather than
directly through the insulin receptor, which also resides within
caveolae(33) .
Alternate initiation from the same mRNA transcript is used to generate two isoforms of another tyrosine kinase substrate: the 52- and 46-kDa isoforms of Shc(34) . In this case, initiation from an internal methionine residue results in an N-terminal 59-amino acid truncation of the 46-kDa isoform relative to the 52-kDa isoform. Like caveolin, these two isoforms are differentially recognized by tyrosine kinases. Specifically, only the 52-kDa isoform is a substrate for the insulin receptor kinase, whereas both the 52- and the 46-kDa isoforms are recognized equally well by the epidermal growth factor receptor kinase (34) . Thus, alternate initiation may provide a means for generating distinct forms of the same gene product that can be differentially regulated by a given signaling molecule.
What could be the functional consequences of the
tyrosine phosphorylation of caveolin ? Caveolin exists as a high
molecular mass oligomer containing 14-16 individual caveolin
molecules(16, 17) , and these caveolin homo-oligomers
have the capacity to self-associate into larger complexes(16) .
Thus, tyrosine-phosphorylated caveolin could potentially serve as an
oligomeric docking site for SH2 domain signaling molecules-much
like activated growth factor receptors that oligomerize, undergo
tyrosine phosphorylation, and recruit SH2 domain-containing proteins to
the cytoplasmic surface of the plasma membrane. We are currently
searching for proteins that preferentially bind to Src-phosphorylated
caveolin.