(Received for publication, September 29, 1994; and in revised form, November 16, 1994)
From the
The ras-GAP associated protein, p62, is a major tyrosine phosphoprotein in transformed and growth factor treated cells. Although its exact function is not known, it can bind directly to src-family tyrosine kinases and has been implicated as a linker protein bridging activated src family tyrosine kinases with downstream effectors. One novel feature of p62, revealed by its predicted amino acid sequence, is the presence of an RNA-binding region, the KH domain. As p62 becomes tyrosine phosphorylated when src-kinases become activated, we compared the RNA binding ability of p62 in both its phosphorylated and unphosphorylated state. The ability of p62 to bind RNA was severely impaired when p62 was tyrosine phosphorylated. This suggests that the ability of p62 to bind RNA is regulated by tyrosine phosphorylation and implicates the regulation of RNA as a component of tyrosine kinase signaling pathways.
p62 is a major tyrosine phosphorylated substrate in growth factor treated and transformed cell lines(1, 2) . Although little is known about its exact function, its ability to be phosphorylated after numerous stimuli and its association with the ras-GTPase activating protein (ras-GAP) suggest that it has an important role in signal transduction, possibly in ras activation(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . The cloning of p62 in 1992, however, did not readily suggest the function of this molecule; analysis of the deduced amino acid sequence only revealed that p62 is related to RNA binding proteins(15) .
Recent studies implicate p62 in cell cycle regulation and tyrosine
kinase mediated signal transduction. p62 (or a related protein) forms
complexes with src during mitosis (16, 17) suggesting
that p62 may play a critical role in cell cycle regulation. Other data
demonstrate that p62 can associate directly with p59 and p60
resulting in tyrosine
phosphorylation of p62(16, 17, 18) . Tyrosine
phosphorylated p62 can bind the SH2 domains of Grb2(19) ,
PLC
-1(19, 20) , and ras-GAP(15) .
However, the function of the p62 RNA binding domain is still not known.
The type of RNA binding domain present in p62 is known as the KH domain (21) . This RNA binding domain is an evolutionarily conserved sequence which has been shown to be integral in the RNA binding ability of FMR1 and hnRNP K(22) . A physiological role for the FMR1 KH domain has been established with the revelation that a single point mutation in a conserved residue of the FMR1 KH domain is associated with severe fragile X syndrome(23, 24) .
The presence of p62, an RNA binding protein, as a prominent tyrosine phosphorylated substrate suggests that some functions of RNA binding proteins may be regulated by tyrosine kinase signaling pathways. The regulation of RNA binding proteins might regulate RNA stability or efficiency of translation, allowing a cell to respond rapidly to external stimuli by directing protein synthesis in the absence of new transcription. How tyrosine phosphorylation might regulate RNA is not known, but one possibility is that phosphorylation regulates binding of RNA. We, therefore, tested the ability of p62 to bind RNA in both its phosphorylated and unphosphorylated state.
Samples were analyzed on 10% SDS-polyacrylamide gels and
transferred to nitrocellulose. Immunoblotting was then performed using
the designated primary antibody followed by goat anti-mouse or
anti-rabbit conjugated to horse radish peroxidase (Organon
Teknika-Cappel) and chemiluminescence was used for protein detection
(DuPont). Anti-myc monoclonal 9E10 (35) was obtained from
American Type Culture Collection. Polyclonal antibody against
p59 was generously provided by André Veillette. 4G10 phosphotyrosine antibody was purchased from Upstate
Biotechnology Inc.
Because there is no physiological RNA target known to bind p62, we utilized homopolymeric RNA for binding assays. This method, first described by Swanson and Dreyfuss(25) , has been used extensively to study protein/RNA interactions(17, 22) . Specifically, we chose poly(U) homopolymer as a binding substrate because it has been previously shown that p68, a p62-related protein, binds this polymeric RNA specifically (17) .
To determine whether tyrosine phosphorylation of p62
affects its ability to bind RNA, an epitope tagged form of p62 was
expressed alone (myc-p62) or co-expressed with p59 in
HeLa cells using the vaccinia T7 expression system(26) . As
reported previously, co-expression of p62 with p59
results in efficient tyrosine phosphorylation of
p62(19) . Cell lysates were divided equally and incubated with
either poly(U) immobilized to agarose or agarose alone. Bound myc-p62
was analyzed by anti-myc immunoblotting (Fig. 1A, lanes 3-6). As expected, poly(U) beads bound p62
efficiently from cell lysates of cells expressing p62 alone (Fig. 1A, lane 4); no binding was detected to
the agarose beads alone (Fig. 1A, lanes 3 and 5). Interestingly, p59
co-expression with p62
resulted in almost no detectable binding of p62 to poly(U) suggesting
that p59
can regulate the ability of p62 to bind RNA (Fig. 1A, lane 6). Inability to detect bound
myc-p62 was not due to poor expression of myc-p62 in this sample;
anti-myc immunoblotting of a portion of the cell extract demonstrated
equivalent expression levels. (Fig. 1A, lanes 1 and 2).
Figure 1:
Effect of
p59 coexpression on p62 binding to RNA. A, coexpression of p59
with p62
abrogates p62/RNA binding. HeLa cell lysates, transfected with either
p62-myc alone (lanes 1, 3, and 4) or
co-transfected with p62-myc and p59
(lanes
2, 5, and 6) were aliquoted and either
immunoblotted (2.5% of lysate) with anti-myc (lanes 1 and 2), or precipitated by an agarose (lanes 3 and 5) or poly(U)-agarose (lanes 4 and 6)
followed by anti-myc immunoblotting. Cell lysates were also
immunoblotted with anti-p59
(lanes 7 and 8). B, p62 binding to RNA is unaffected by
mixing lysates of p59
and p62 in vitro.
p62-myc or p59
were expressed in separate HeLa
cell cultures. Lysates were mixed at various stoichiometries before
binding reaction and anti-myc immunoblotting. p62 association with
agarose is shown in lane 1. p59
lysate was added
to a constant amount of p62 lysate before poly(U) binding at the
proportion of p62 lysate indicated in the figure label above each
respective lane (lanes 2-5).
As p62 and p59 can form stable
complexes, one possible explanation for this result was p59
competition with p62 for poly(U) binding. A lysate mixing
experiment was done to rule out this possibility. p62 or p59
were expressed independently in separate HeLa cell cultures, and
extracts were mixed and preincubated prior to binding studies. Fig. 1B shows that the addition of increasing amounts
of p59
cell lysate had no affect on binding of p62
binding to poly(U) (Fig. 1B, lanes 2-5).
This argues that an in vivo interaction between p62 and
p59
is necessary for loss of p62/RNA binding.
We were
next interested in defining the nature of the p62/p59 interaction required for the abrogation of p62 binding to RNA. To
assess the role of phosphorylation in p62/RNA binding, p62 was
co-expressed with a kinase inactive form of p59
(Dfyn).
HeLa cells were co-transfected with cDNAs for p62 and
p59
, p62 and Dfyn or with p62 alone. Co-expression of
kinase inactive p59
did not affect the interaction of p62
with RNA (lane 9). Immunoblotting of whole cell lysates were
performed to confirm p62 and p59
expression levels in the
appropriate transfected cells (Fig. 2, lanes
10-12). The level of endogenous p59
is shown
in lane 10, while lanes 11 and 12 confirmed
increased quantities of p59
in the co-transfections.
Anti-phosphotyrosine immunoblotting was performed on transfected cell
lysates to confirm that Dfyn was, in fact, inactive and that
co-expression of p59
with p62 resulted in tyrosine
phosphorylation of p62 (Fig. 2, lanes 13-15).
This concordance between phosphorylated p62 levels and diminution of
p62/RNA interaction implicates phosphorylation as a key regulatory
element in p62's propensity to bind RNA.
Figure 2:
Phosphorylation of p62 by
p59determines its ability to bind RNA. A, inhibition of p62 binding to RNA by p59
is dependent on p59
kinase activity.
Binding assays were performed from cell lysates expressing p62-myc
alone (lanes 1, 4, and 5), or co-expressing
p62-myc and p59
(lanes 2, 6,
and 7), or co-expressing p62-myc and kinase inactive
p59
(Dfyn, lanes 3, 8, and 9). p62 binding to agarose is shown in lanes 4, 6, and 8; p62 binding to poly(U) is shown in lanes 5, 7, and 9. To confirm expression of
p62 and p59
, a portion of the lysates were
immunoblotted with antibodies to myc (lanes 1-3),
p59
(lanes 10-12) and
phosphotyrosine (lanes 13-15) B, phosphatase
treatment of phosphorylated p62 rescues its ability to bind RNA.
Lysates from cells expressing p62-myc alone (lanes 1, 3, 4, and 5) or cotransfected with
p59
(lanes 2 and 6-8)
were aliquoted and incubated for 30 min (room temperature) in the
presence (+) or absence(-) of calf intestinal phosphatase.
Binding studies using either agarose (lanes 3 and 6)
or poly(U) (lanes 4, 5, 7, and 8)
were performed followed by anti-myc immunoblotting. Equivalent levels
of p62 expression was confirmed by myc immunoblotting (lanes 1 and 2). To confirm the phosphatase treatment, a portion
of the lysate was immunoblotted with antibodies to phosphotyrosine (lanes 9-12).
Dephosphorylation of
p62 should therefore rescue the ability of p62 to bind RNA. Expression
of p62 (with or without p59 coexpression) and binding to
poly(U) were performed as described previously with one modification:
prior to the binding reaction, lysates were incubated for 30 min with
CIP. As previously shown, under normal binding conditions (no CIP), p62
bound RNA (Fig. 2B, lane 4), an interaction
which was impaired by coexpression of p59
(Fig. 2B, lane 7). Strikingly,
dephosphorylation of p62 (subsequent to p59
-induced
phosphorylation) rescued p62/RNA binding (Fig. 2B, lane 8). Phosphatase treatment of lysates from cells
expression p62 alone left p62 binding to RNA relatively unaffected (Fig. 2B, lane 5). Anti-myc immunoblotting of
whole cell lysates confirmed equivalent expression levels of p62 (Fig. 2B, lanes 1 and 2), and
anti-phosphotyrosine immunoblotting confirmed p62 dephosphorylation
under phosphatase conditions (Fig. 2B, lanes
9-12).
It has been shown that the sites of tyrosine
phosphorylation by p59 and src reside in the
carboxyl-terminal region of p62 (15, 19) . To test the
involvement of the carboxyl-terminal region of p62 in regulation of RNA
binding, we transfected a p62 construct lacking the tyrosine rich
carboxyl-tail (p1-4(19) ) and compared its binding activity
after co-expression with p59
. Binding studies were done,
as described previously, followed by anti-myc immunoblotting (Fig. 3, lanes 3-6). There was little RNA binding
difference between p1-4 alone and p1-4 co-expressed with p59
both formed strong associations with poly(U). Anti-myc and
anti-p59
immunoblotting were performed to confirm
equivalent and appropriate expression levels of p62 and p59
(Fig. 3, lanes 1 and 2 and data not
shown). Therefore, tyrosine phosphorylation of the carboxyl-terminal
region of p62 is involved in the regulation of p62/RNA binding.
Figure 3:
Regulatory tyrosines of RNA binding map to
the tyrosine rich C terminus of p62. p62-myc, with C-terminal
truncation of the tyrosine rich tail (p1-4), was expressed alone (lanes 1, 3, 4), or cotransfected with
p59 (lanes 2, 5, and 6) in HeLa cells. Anti-myc immunoblotting was performed on
2.5% of each lysate (lanes 1 and 2) and on portions
of lysate passed over agarose (lanes 3 and 5) or
poly(U) column (lanes 4 and 6).
We show here that p62 binding to RNA is highly dependent on the tyrosine phosphorylation state of p62, with only unphosphorylated p62 able to interact with RNA. Furthermore, we mapped the negative regulatory tyrosines, necessary for phosphorylation-mediated inhibition of p62/RNA binding, to the carboxyl-terminal region of p62. This region, however, does not directly mediate p62 binding to RNA by virtue of previously published reports(15) , and also by our observation that truncation of the carboxyl-terminal region does not abrogate p62/RNA interaction (Fig. 3). The negative regulatory tyrosines in the carboxyl-terminal domain of p62 must therefore indirectly mediate the loss of p62 binding to RNA. One possibility is that p62 only binds to RNA as an oligomer whose formation is inhibited by tyrosine phosphorylation. This may explain how the single KH domain in p62 is capable of binding RNA while another KH homology protein, FMR1, requires multiple tandem KH domains to bind RNA(21, 22, 27, 28) . Another possibility is that the phosphorylated carboxyl terminus which is negatively charged interacts with the RNA contact site of the KH domain thus sterically inhibiting the interaction of p62 with RNA. We are currently testing both of these possibilities.
In addition to the KH domain, p62 contains another RNA binding motif, an RGG box. This motif was originally shown to confer RNA binding to hnRNP U protein(29) . It is characterized by several closely spaced arginine-glycine-glycine (RGG) repeats (6 to 25) usually with intervening aromatic residues. The RGG box within the p62 sequence is abbreviated, comprised of only two RGG sequences, amino-terminal to the KH domain. However, the p62 construct used for our assays did not include this RGG box. Because this protein can still bind RNA, the ability of p62 to bind RNA is not dependent on this sequence. We cannot rule out, however, that this motif does not play any role in the regulation or specificity of RNA binding.
The finding that tyrosine phosphorylation regulates p62 binding to RNA is remarkable because it suggests for the first time that tyrosine kinase signaling pathways are involved in the regulation of RNA. The regulation of RNA by signaling could allow a quiescent cell to respond very rapidly to external stimuli, much faster than protein expression from de novo transcription. As many cellular responses to signaling are rapid (within minutes), it is surprising that most signaling research has focused on the regulation of new transcription by signaling pathways rather than the regulation of stored RNAs. Such regulation might be envisioned to occur either by enhancing mRNA stability(30, 31) , by regulating the rate and efficiency of mRNA translation (32) or by specific targeting of mRNA in the cytoplasm (33) . Specific localization of mRNAs in the cell could play an important role in the delivery of specific proteins to specific structures. As many cytoskeletal proteins contain SH3 domains, one possibility is that p62/p68 helps target RNA to cytoskeletal structures via SH3-mediated interactions. The finding that protein/RNA interactions are regulated by tyrosine phosphorylation suggests that RNA binding proteins will play specific and important roles during signal transduction, cell growth and differentiation.