hnRNP K specifically binds and activates Src kinase in vivo and in vitro.
Since activated c-Src and Lck can phosphorylate hnRNP K in vitro (19
), we wanted to examine whether hnRNP K can be phosphorylated by active forms of Src kinase in transfected cells. HeLa cells were cotransfected with expression vectors for wild-type c-Src, constitutively active or inactive c-Src mutants, and histidine-tagged hnRNP K. Protein expression was monitored by Western blotting of total cell lysates using antibodies directed against the His tag (Fig. ), c-Src (Fig. ), phosphotyrosine (Fig. ), and c-Src phosphorylated at Tyr-416 (Fig. ), which indicates Src kinase activity. Surprisingly, transfection of wild-type c-Src with His-hnRNP K (Fig. , compare lanes 1 and 4) causes an increase in tyrosine phosphorylation of cellular proteins (Fig. , lanes 1 and 4). This increase in tyrosine phosphorylation reflects posttranslational Src kinase activation over the basal activity (Fig. , compare lanes 1 and 2 with lane 4), because the levels of c-Src protein are similar (Fig. , lanes 1, 2, and 4). As a positive control for the activated state of c-Src, we transfected 5 μg of His-hnRNP K together with the constitutively active mutant Src-KP or Src-Y527F (8
) (Fig. , lanes 5 and 6). The overall tyrosine phosphorylation of cellular proteins with 5 μg of His-hnRNP K and wild-type c-Src is at least as high as the levels observed with the activated mutants (Fig. , compare lanes 4, 5, and 6). By contrast, the inactive autophosphorylation site mutant Src-Y416F fails to be activated by hnRNP K and does not increase cellular phosphotyrosine levels following the cotransfection of hnRNP K (Fig. , lane 7). These findings suggest that hnRNP K is able to activate the tyrosine kinase activity of c-Src in vivo.
To test for a direct interaction of hnRNP K and different forms of Src in vivo, His-hnRNP K was precipitated with a His antibody. The immunoprecipitates were subsequently analyzed by Western blotting (Fig. ). As is evident from Fig. , both the tyrosine-phosphorylated and the nonphosphorylated forms of hnRNP K interact with c-Src and the Src mutants (lanes 4 to 7). Thus, following cotransfection of hnRNP K and the wild type c-Src kinase into HeLa cells, the two proteins interact with each other, c-Src kinase is activated, and hnRNP K and other proteins become tyrosine phosphorylated. As is evident from the inactive Src(Y416F) mutant (Fig. , lane 7), Src activity and hnRNP K phosphorylation are not required for the interaction.
We next assessed whether the ability to interact with and activate c-Src was shared by hnRNP E1, another KH domain RNA binding protein which together with hnRNP K binds to the DICE and regulates LOX expression (16
). hnRNP E1 can bind only minor quantities of c-Src (Fig. ), is very weakly phosphorylated (Fig. ), and completely fails to activate c-Src in vivo (Fig. , compare lanes 1 and 2 with lane 4). We conclude that the ability to interact with and activate c-Src is a specific feature of hnRNP K. We also found that the tyrosine kinase c-Abl, the cellular homologue of the transforming gene of Abelson murine leukemia virus, fails to associate with and be specifically activated by hnRNP K (data not shown).
hnRNP K is an RNA-binding protein without any domains that resemble protein kinases or protein phosphatases that could help explain the Src activation. To distinguish whether the activation of c-Src by hnRNP K is direct or indirect, we next tested whether recombinant hnRNP K could activate purified c-Src in vitro. Wild-type c-Src and the constitutively active Y527F mutant as a positive control were immunopurified from transfected human HEK-293 cells and incubated with the Src substrate enolase in the presence of [γ-32P]ATP. The basal phosphorylation of enolase (Fig. , lanes 2) is stimulated approximately fivefold by replacement of wild-type c-Src with the active Y527F mutant (lanes 1). Addition of recombinant hnRNP K to wild-type c-Src stimulates enolase phosphorylation in a dose-dependent manner up to sixfold (Fig. , lanes 3 and 4). The activation of c-Src by hnRNP K is inhibited by the selective inhibitor of Src kinase PP2 (Fig. , lanes 5) but not by PP3 (lanes 6), which was included as a negative control. By contrast to hnRNP K and in keeping with the in vivo data, recombinant hnRNP E1 (Fig. , lanes 7 and 8) fails to activate c-Src. We conclude that hnRNP K specifically activates c-Src in vivo and in vitro and that this activation is likely to be direct. c-Src, activated by hnRNP K, can phosphorylate both hnRNP K and an external substrate, enolase.
FIG. 2. hnRNP K but not hnRNP E1 activates Src catalytic activity in vitro. Wild-type Src (wt) and the constitutively active Src(Y527F) mutant as a positive control were immunopurified from transfected human 293 HEK cells, and equal amounts were assayed with (more ...) The SH3 domain of c-Src is important for interaction with hnRNP K.
A peptide corresponding to the SH3 domain of c-Src has been shown to interact with the proline-rich domain of hnRNP K in vitro (23
). To evaluate the importance of this region for the binding of c-Src to hnRNP K in HeLa cells, we compared His-hnRNP K binding to c-Src with Src mutants lacking either its SH3 or its SH2 domain, Src(−SH3) and Src(−SH2), respectively, by coimmunoprecipitation with antihistidine (Fig. ) or anti-Src (Fig. ) antibodies. In contrast to c-Src and Src(−SH2), the Src(−SH3) mutant (Fig. , lanes 2) displays a much reduced interaction with hnRNP K (Fig. ) and yields barely detectable levels of tyrosine phosphorylation of hnRNP K (Fig. ). Hence, the SH3 domain of Src is important for both the binding and the phosphorylation of hnRNP K, but a weak interaction between the two proteins can also be established in the absence of the SH3 domain.
Multiple tyrosine residues in hnRNP K are targets of Src.
Earlier analyses of hnRNP K predicted four tyrosine residues as targets of Src (Tyr 230, 234, 236, and 380) (22
), and Tyr 230, 234, and 236 have been shown to contribute to hnRNP K phosphorylation by Src in vitro (19
). In initial experiments in HeLa cells, we found that the hnRNP K mutant in which the tyrosine residues 230, 234, and 236 were changed to phenylalanine (called Y3F) was still phosphorylated by c-Src (data not shown). A Y4F hnRNP K mutant (with the sites 230, 234, 236, and 380 changed) can bind both wild-type c-Src and the inactive Src mutant Src(Y416F) (Fig. , lanes 1 to 6). Y4F is also still phosphorylated by c-Src (Fig. , lane 5), although at a lower level (81%) than wild-type hnRNP K (compare lane 2). In order to identify the tyrosine phosphorylation sites of hnRNP K targeted by c-Src in vivo, phosphopeptide mapping by mass spectrometry was carried out. By using parent ion scans, which provide a selective detection method for phosphopeptides (14
), 6 phosphorylated tyrosines out of the 17 tyrosine residues present in hnRNP K (Fig. , top) were determined. We confirmed that Tyr 230, 234, 236, and 380 are phosphorylated and identified two additional targets of c-Src, Tyr 72 and Tyr 225 (data not shown).
Based on this result, the appropriate Y6F hnRNP K mutant was generated and tested. Tyrosine phosphorylation of hnRNP K (Y6F) was further reduced compared to that of both hnRNP K wild-type (32%) and hnRNP K (Y4F) (41%) in HeLa cells cotransfected with c-Src (Fig. , lanes 2, 5, and 8). No hnRNP K phosphorylation was observed following cotransfection with the negative control Src(Y416F) (lanes 3, 6, and 9). Nonetheless, even the Y6F mutant still interacted with and was phosphorylated by c-Src (Fig. , lanes 8). These findings indicate that Y 72, 225, 230, 234, 236, and 380 contribute to phosphorylation by c-Src but that additional tyrosine residues of hnRNP K are targeted by the kinase.
Attempts to map additional tyrosine residues in the hnRNP K Y6F mutant phosphorylated by c-Src by mass spectrometry were challenged by insufficiently low signal-to-noise ratios.
Tyrosine phosphorylation of hnRNP K by c-Src reversibly inhibits DICE binding.
To directly examine the effect of Src-mediated phosphorylation on the binding of hnRNP K to the DICE, His-tagged recombinant human hnRNP K was incubated with immunopurified c-Src, the activated mutant Src (KP), the inactive mutant Src(Y416F), or phosphorylation buffer without the kinase (Fig. ). Subsequently, successful and specific tyrosine phosphorylation of hnRNP K was confirmed with an antiphosphotyrosine antibody (Fig. ). The phosphorylation of hnRNP K by c-Src in vitro causes a small reduction of its electrophoretic mobility compared to the nonphosphorylated protein (Fig. , compare lanes 2 and 4 with lanes 1 and 3). Importantly, a Northwestern blot with a radioactively labeled DICE shows that the binding of hnRNP K to the DICE is drastically reduced after the phosphorylation by c-Src kinase (Fig. ). Subsequent treatment with λ-phosphatase reverses both the tyrosine phosphorylation and the inability of hnRNP K to bind to the DICE probe (Fig. , lanes 3 and 5). As predicted by the data in Fig. and , Src does not affect the ability of hnRNP E1 to bind to the DICE (Fig. ). These results show that the tyrosine phosphorylation of hnRNP K by c-Src permits the specific and reversible regulation of the binding of hnRNP K to its physiological binding site, the DICE.
c-Src rescues translation inhibited by hnRNP K in vivo.
Cotransfection of hnRNP K with a LUC reporter mRNA bearing a DICE in its 3′ UTR (LUC-DICE) recapitulates LOX silencing and causes a specific, DICE-dependent reduction of LUC activity in transfected HeLa cells. The stability of the LUC reporter mRNA is not affected by coexpressed hnRNP K (16
) (data not shown). For use as a negative control mRNA that is not silenced by hnRNP K, the DICE was replaced in LUC-NR by NR sequences of the LOX mRNA 3′ UTR (16
). To investigate whether hnRNP K phosphorylation by c-Src controls its activity as a repressor of LUC-DICE mRNA translation in vivo, HeLa cells were transfected with LUC-DICE or LUC-NR reporter constructs (Fig. ). These LUC indicator constructs were cotransfected with an hnRNP K expression vector (or an expression vector for the RNA binding protein U1A as a specificity control) and different forms of c-Src (Fig. ). As expected, LUC activity from LUC-DICE, but not LUC-NR, is inhibited by hnRNP K. This inhibition is relieved by coexpression of wild type c-Src or of the constitutively active Src-KP (K249E, P250E) mutant. By contrast, cotransfection of the inactive mutant Src-Y416F fails to stimulate LUC activity. This shows that a Src mutant that binds hnRNP K (Fig. ) but fails to phosphorylate it is insufficient to activate LUC-DICE translation. LUC-NR expression is not affected by hnRNP K alone or in combination with any of the Src mutants, demonstrating the specificity of the observed effects for the hnRNP K/DICE interaction.
To further evaluate the role of c-Src in the activation of LUC-DICE expression, we used the potent and selective inhibitor of Src kinase PP2, or PP3 as a negative control (11
). Treatment of transfected HeLa cells with PP2, but not PP3, abolishes the stimulatory effect of c-Src or Src-KP on LUC-DICE expression (Fig. ). This pharmacological effect of PP2 is associated with a lack of hnRNP K phosphorylation but not with a failure to express the hnRNP K protein (Fig. ). These data demonstrate that tyrosine phosphorylation of hnRNP K by c-Src can regulate the function of the hnRNP K/DICE mRNA silencing system in vivo.