(Received for publication, January 19, 1996; and in revised form, March 15, 1996)
From the
Lck, a lymphocyte-specific tyrosine protein kinase, is bound to
cellular membranes as the result of myristoylation and palmitoylation
of its amino terminus. Its activity is inhibited by phosphorylation of
tyrosine 505 and stimulated by phosphorylation of tyrosine 394. The
Tyr-505 Phe mutant of Lck (F505Lck) exhibits elevated biological
activity and constitutive phosphorylation of Tyr-394 in vivo.
Mutations at sites of fatty acylation that prevent F505Lck from
associating with cellular membranes abolish the biological activity of
the molecule in vivo, compromise its activity as a protein
kinase in vivo and in vitro, and eliminate the
phosphorylation of Tyr-394. Here, we show that exposure of cells
expressing cytoplasmic or nuclear forms of F505Lck to
H
O
, a general inhibitor of tyrosine protein
phosphatases, restores the catalytic activity of these mutant proteins
through stimulation of phosphorylation of Tyr-394. H
O
treatment induced the phosphorylation of Tyr-394 on catalytically
inactive forms of Lck regardless of cellular localization.
Phosphorylation of Tyr-394 therefore need not occur by
autophosphorylation. Thus, there appear to be two mechanisms through
which the phosphorylation of Lck at Tyr-394 can occur. One is
restricted to the plasma membrane and does not require the presence of
oxidants. The other is operational in the nucleus as well as the
cytosol and is responsive to oxidants.
p56, a member of the Src family of
tyrosine protein kinases(1, 2) , is expressed
exclusively in lymphoid cells and predominantly in T cells. Lck
associates with the cytoplasmic domain of the T-cell
receptor-associated glycoproteins CD4 and CD8(3, 4) ,
membrane immunoglobulin in B cells(5) , and
glycosylphosphatidylinositol-anchored proteins (6, 7) through its unique amino terminus.
p56
is essential for both T-cell development (8, 9) and T-cell
activation(10, 11) . The activity of Lck is regulated
by the phosphorylation of tyrosine residues at positions 394 and 505.
Phosphorylation of Tyr-505 inhibits the activity of Lck (12, 13) . Mutation of Tyr-505 to phenylalanine
results in a constitutively active protein that is able to transform
fibroblasts (12, 13) , enhance T-cell responsiveness
to antigen(14) , and induce antigen-independent interleukin-2
production in T cells(15) . In contrast, phosphorylation of
Tyr-394, the single site of autophosphorylation in vitro,
enhances the catalytic activity of Lck and is required for the
activation of Lck by mutation of Tyr-505 to
phenylalanine(16, 17, 18) .
Lck associates with the inner face of the plasma membrane through its amino terminus. This interaction is mediated by both myristic acid and palmitic acid that are bound to the amino-terminal glycine and Cys-3 and/or Cys-5, respectively(19, 20, 21, 22) . Mutations in which the amino-terminal glycine is mutated to alanine result in a protein that is neither myristoylated nor palmitoylated and is unable to associate with the plasma membrane(17) . Mutation of both Cys-3 and Cys-5 to serine yields a protein that is myristoylated but not palmitoylated (21, 22, 23) . The resulting Lck protein is completely cytoplasmic, indicating that myristoylation is not sufficient for anchoring Lck to the plasma membrane(22) .
Hydrogen peroxide is a general inhibitor of tyrosine protein
phosphatases(24) . Exposure of cells to HO
induces rapid tyrosine phosphorylation of numerous cellular
proteins in vivo(24, 25, 26, 27, 28) and
has been shown to be a potent activator of Lck(18) . Activation
of Lck by H
O
is due to an increase in the
phosphorylation of Tyr-394(18) . In the current study, we
examined the effect of H
O
on three different
non-membrane-bound forms of genetically activated Lck (F505Lck).
Non-myristoylated, myristoylated but non-palmitoylated, and nuclear
forms of F505Lck all exhibit severely reduced activity in vivo and in vitro. H
O
is able to
restore the in vitro catalytic activity of all three
non-membrane-bound forms of Lck. This increase in activity correlates
with, and in all probability is due to, an increase in the
phosphorylation of Tyr-394. Apparently, phosphorylation of Tyr-394
occurs inefficiently when Lck is located at sites other than cellular
membranes but can be induced by elevated levels of oxidants.
Figure 1:
Intracellular
location of mutant forms of Lck in fibroblasts. Rat 208F fibroblasts
expressing mutant Lck proteins were permeabilized and incubated
sequentially with a rabbit polyclonal antisera against Lck and a goat
anti-rabbit immunoglobulin secondary antibody conjugated to fluorescein
isothiocyanate. Bound antibody was visualized on a Zeiss Axiophot
fluorescence microscope using a 63 objective. A,
uninfected 208F; B, wild-type Lck; C, S3/5F505Lck; D, nucLck; E, nucF505Lck; F, F505Lck; G, F505Lck +H
O
; H,
A2F505Lck; I, A2F505Lck
+H
O
.
Figure 2:
Tyrosine protein phosphorylation in cells
expressing non-membrane-associated F505Lck. A,
anti-phosphotyrosine Western blot. Lysates of fibroblasts expressing
mutant Lcks were prepared and fractionated by SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to a polyvinylidene
difluoride membrane and incubated with antibodies against
phosphotyrosine and I-protein A. Lane 1,
uninfected 208F cells; lane 2, wild-type Lck (WTLck); lane 3, F505Lck; lane 4, nucF505Lck; lane 5,
S3/5F505Lck; lane 6, A2F505Lck. B, anti-p56lck
Western blot. Lck immunoprecipitates from fibroblasts expressing mutant
Lcks were fractionated by SDS-polyacrylamide gel electrophoresis,
transferred to a polyvinylidene difluoride membrane, and incubated with
anti-Lck antibodies and
I-protein A. The identity of the
lanes is the same as in A. Non-membrane-bound forms of Lck
have reduced electrophoretic mobility and typically exhibit multiple
apparent molecular weights.
Figure 3:
In vitro protein kinase activity
of non-membrane-bound forms of F505Lck. Mutant Lcks were isolated by
immunoprecipitation and assayed in vitro for their ability to
phosphorylate an exogenous substrate, Val5-angiotensin II. Assay
results are expressed as the rate of labeled phosphate incorporation by
the substrate per arbitrary unit of Lck. For normalization, Lck protein
levels in the samples were measured by Western blotting of a fraction
of the immunoprecipitates with antibodies to Lck and I-protein A. A, relative activities of wild-type (WT) Lck, F505Lck, nucF505Lck, S3/5Lck, and A2F505Lck from
unstimulated cells. B, activities of non-membrane-bound forms
of F505Lck before and after exposure to
H
O
.
Figure 4:
Analysis of Lck phosphorylation by
two-dimensional tryptic peptide mapping. Lck was isolated by
immunoprecipitation from cells that had been labeled biosynthetically
with P
. Lck was purified by SDS-polyacrylamide
gel electrophoresis, transferred to nitrocellulose, and digested with
trypsin. The resulting peptides were separated horizontally by
electrophoresis at pH 8.9 and then vertically by ascending
chromatography. The peptides containing Tyr-394 are marked with open arrowheads. The sample origins are indicated by filled arrowheads. A, nucF505Lck; B,
S3/5F505Lck; C, A2F505Lck; D, nucF505Lck
+H
O
; E, S3/5F505Lck
+H
O
; F, A2F505Lck
+H
O
.
Figure 5:
The effect of HO
treatment on the phosphorylation of catalytically inactive,
non-membrane-associated Lck. Cells were labeled biosynthetically with
P
and then exposed to H
O
as described. Two-dimensional tryptic peptide mapping was
performed as in Fig. 4. Sample origins are indicated by arrowheads. A, nucR273Lck from unstimulated
fibroblasts; B, nucR273Lck from fibroblasts stimulated with
H
O
; C, A2R273Lck from unstimulated
fibroblasts; D, A2R273Lck from fibroblasts stimulated with
H
O
.
The lack of in vivo catalytic and biological
activity of A2F505Lck, S3/5F505Lck, and nucF505Lck, none of which can
bind stably to cellular membranes, shows that F505Lck must be able to
associate with membranes to be active. All of these
non-membrane-associated forms of F505Lck differ from F505Lck in that
they lack detectable phosphorylation on Tyr-394, a residue at which
phosphorylation positively regulates Lck and that is highly
phosphorylated in membrane-bound F505Lck. It is likely that this lack
of phosphorylation in vivo and in vitro is
responsible for their low
activity(17, 18, 39) . This implies that
there is some protein or cofactor localized to membranes that is
normally required for the activation of F505Lck through phosphorylation
of Tyr-394. Phosphorylation of Tyr-394, however, can occur at
intracellular sites other than the cellular membranes. When cells
expressing cytoplasmic or nuclear F505Lck are exposed to
HO
, both the phosphorylation of Tyr-394 and the in vitro catalytic activity of the mutant Lcks are stimulated
markedly. This rules out the possibility that intrinsic defects in the
mutagenized genes encoding nucF505Lck, S3/5F505Lck, and A2F505Lck are
responsible for the lack of activity in these proteins in unstimulated
cells. However, the mechanism by which phosphorylation of Tyr-394 in
the cytosol and nucleus occurs is unclear. It is not a result of
relocalization to the plasma membrane. Immunofluorescence microscopy (Fig. 1, panels H and I) as well as cell
fractionation experiments (data not shown) show clearly that the bulk
of A2F505Lck remains cytosolic in H
O
-treated
cells. While a small population of A2F505Lck may relocalize to the
plasma membrane in the presence of H
O
, the
extensive phosphorylation of A2F505Lck on Tyr-394 indicates that
reorganization of cellular location is not the mechanism by which the
kinase is activated.
The phosphorylation of A2F505Lck and nucF505Lck
is also not necessarily the result of autophosphorylation since
catalytically inactive A2Lck and nucLck become phosphorylated at
Tyr-394 when cells lacking active Lck are treated with
HO
. There must, therefore, be at least one
tyrosine kinase in the cytoplasm and nucleus that has the ability to
phosphorylate Lck at Tyr-394. This unidentified kinase may normally be
subject to regulation by oxidant levels within the cell. In addition,
apparently undiminished phosphorylation of Tyr-505 was observed in
cytosolic and nuclear-targeted forms of kinase-inactive Lck. The
tyrosine protein kinase CSK phosphorylates the carboxyl-terminal
tyrosine of Src family members, including Tyr-505 of Lck, and
negatively regulates the activities of these proteins(40) . Our
results demonstrate that Csk is able to phosphorylate Tyr-505 of
non-membrane-bound, kinase-inactive Lck. The fact that Tyr-505 was
phosphorylated in non-membrane-bound forms of kinase-inactive Lck
suggests that either CSK itself or a CSK-like activity is present in
the nucleus as well as the cytosol.
S3/5F505Lck and A2F505Lck, which cannot be phosphorylated at position 505, are less active as tyrosine kinases in vitro than S3/5Lck or A2Lck, which contain phosphorylated Tyr-505 (data not shown). This difference may result from the fact that the SH2 domain of A2F505Lck and S3/5F505Lck does not have a phosphorylated Tyr-505 with which to form an intramolecular complex. In wild-type Lck, the SH2 domain binds to phosphorylated Tyr-505 (41) and apparently yields a conformation that exhibits in vitro activity in the absence of extensive phosphorylation at Tyr-394. In contrast, the untethered SH2 domain of F505Lck may induce a conformation that requires phosphorylation of Tyr-394 for catalytic activity.
The properties of nucF505Lck, S3/5F505Lck, and A2F505Lck differ from those of analogous cytosolic forms of the activated Src kinase. A2v-Src and A2F527c-Src, although nontransforming when expressed in fibroblasts, both retain the ability to phosphorylate most cellular substrates in vivo, and A2v-Src exhibits undiminished activity to phosphorylate exogenous substrates in vitro(42, 43) . The reason for the differences between the cytosolic, non-membrane-bound forms of activated Lck and Src is not clear.
It appears that there are at least two activities
that can phosphorylate Tyr-394. One is restricted to the plasma
membrane and does not require the presence of oxidants for activity.
Most likely, this is the mechanism that is responsible for the high
level of Tyr-394 phosphorylation and increased activity of F505Lck at
the plasma membrane. This membrane-restricted activity could be due to
Lck itself or another Src family member. Additionally, there are one or
more activities in the cytosol and nucleus that can phosphorylate
Tyr-394, but these kinases require activation by oxidants. It is
unlikely that a Src family kinase is responsible for this
phosphorylation since Src family proteins are generally associated with
cellular membranes. In the absence of oxidants, the lack of
phosphorylation at Tyr-394 in forms of F505Lck not tethered to
membranes could result from simple lack of phosphorylation of this site
or a more rapid rate of dephosphorylation than phosphorylation of the
site. Since HO
will almost certainly inhibit
the phosphatase responsible for dephosphorylating Tyr-394 in the
cytosol, we cannot determine whether the phosphorylation of Tyr-394
induced by H
O
results from activation of the
kinase phosphorylating the site or simply from inhibition of the
phosphatase dephosphorylating the site. Nevertheless, it is clear that
the steady-state phosphorylation of Tyr-394 in F505Lck is greatly
reduced when the protein is removed from membranes and that there must
exist one or more enzymes or functions restricted to membranes that
facilitate constitutive phosphorylation of Tyr-394. An important
question is the identity of the normal substrates of the
oxidant-regulated tyrosine protein kinases in the cytosol and nucleus
and the role of these tyrosine protein kinases in normal signal
transduction.