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. 2007 Mar;27(5):1758-70.
doi: 10.1128/MCB.02014-06. Epub 2006 Dec 18.

Deciphering the cross talk between hnRNP K and c-Src: the c-Src activation domain in hnRNP K is distinct from a second interaction site

Dörte Adolph  1 , Nadine Flach, Katharina Mueller, Dirk H Ostareck, Antje Ostareck-Lederer
Affiliations

Deciphering the cross talk between hnRNP K and c-Src: the c-Src activation domain in hnRNP K is distinct from a second interaction site

Dörte Adolph et al. Mol Cell Biol. 2007 Mar.

Abstract

The protein tyrosine kinase c-Src is regulated by two intramolecular interactions. The repressed state is achieved through the interaction of the Src homology 2 (SH2) domain with the phosphorylated C-terminal tail and the association of the SH3 domain with a polyproline type II helix formed by the linker region between SH2 and the kinase domain. hnRNP K, the founding member of the KH domain protein family, is involved in chromatin remodeling, regulation of transcription, and translation of specific mRNAs and is a target in different signal transduction pathways. In particular, it functions as a specific activator and a substrate of the tyrosine kinase c-Src. Here we address the question how hnRNP K interacts with and activates c-Src. We define the proline residues in hnRNP K in the proline-rich motifs P2 (amino acids [aa] 285 to 297) and P3 (aa 303 to 318), which are necessary and sufficient for the specific activation of c-Src, and we dissect the amino acid sequence (aa 216 to 226) of hnRNP K that mediates a second interaction with c-Src. Our findings indicate that the interaction with c-Src and the activation of the kinase are separable functions of hnRNP K. hnRNP K acts as a scaffold protein that integrates signaling cascades by facilitating the cross talk between kinases and factors that mediate nucleic acid-directed processes.

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Figures

FIG. 1.
FIG. 1.
Sequence alignment of human hnRNP K and hnRNP E1. The positions of KH domains 1, 2, and 3 are indicated with red bars. The proline (pro)-rich motifs are underlined in black. The c-Src phosphorylation site of hnRNP K that affects DICE binding is indicated by a red arrow (29), and the tyrosine residues identified as targets of c-Src by mass spectrometry are marked with blue arrows (39). The classification of the individual proline-rich motifs as class I or class II according to their orientation in the context of the bound SH3 domain is indicated in red or green, respectively.
FIG. 2.
FIG. 2.
hnRNP K(ΔP1-3) fails to activate, but still interacts with, c-Src. (A) HeLa cells were transiently cotransfected with LUC-DICE cDNA (lanes 1 to 7), with cDNAs coding for U1A as a specificity control (lane 1), His-hnRNP K (lanes 2 to 4), or His-hnRNP K(ΔP1-3) (lanes 5 to 7), with Src(wt) (lanes 3 and 6) or the inactive variant Src(Y416F) (lanes 4 and 7), or without cDNAs (lane 8). HeLa cell lysate was resolved by SDS-PAGE and analyzed in Western blot assays with antibodies against the His tag (His-ab), Src (Src-ab), and GAPDH (GAPDH-ab) as a loading control. His-tagged hnRNP K and His-hnRNP K(ΔP1-3) were immunoprecipitated with an anti-His antibody, resolved by SDS-PAGE, and analyzed by Western blot assays with antibodies against the His tag (His-ab), Src (Src-ab), or p-Tyr (pY-ab). Peptides recognized by the pY-ab nonspecifically are marked as nonspecific (n.s.). (B) LUC activity assay showing the relative (rel.) enzymatic activity levels of the expressed LUC protein.
FIG. 3.
FIG. 3.
The proline motifs P2 and P3 of hnRNP K are responsible for c-Src activation in vitro. The inactive c-Src variant Src(Y416F) (lane 2), the constitutively active form Src(KP) (lane 3), and Src(wt) (lanes 4 to 12) were immunopurified from transfected HeLa cells. Equal amounts, as shown in a Western blot assay with a Src-ab, were incubated in the presence of ATP with equal amounts of recombinant His-hnRNP K (lanes 1 to 4) or its Pro-Ala variants (lanes 5 to 11), as shown in a Western blot assay with an anti His-antibody (His-ab). Phosphorylation of His-hnRNP K or its variants was analyzed in a Western blot assay with a pY-ab. wt, wild type.
FIG. 4.
FIG. 4.
Proline motifs P2 and P3 of hnRNP K are responsible for c-Src activation in vivo. (Left) HeLa cells were cotransfected with cDNAs coding for His-hnRNP K (lanes 1 to 4) or its Pro-Ala variants (lanes 5 to 11), the inactive form Src(Y416F) (lane 2), the constitutively active form Src(KP) (lane 3), or Src(wt) (lanes 4 to 11). (A) HeLa cell lysate was resolved by SDS-PAGE and analyzed in Western blot assays with antibodies against the His tag [His-ab], Src [Src-ab], Src phosphorylated at Y416 [Src(pY416)-ab], and GAPDH [GAPDH-ab] as a loading control. (B) His-tagged hnRNP K or its variants were immunoprecipitated with an anti-His antibody, resolved by SDS-PAGE, and analyzed by Western blot assays with antibodies against the His tag (His-ab), p-Tyr (pY-ab), and Src (Src-ab). (Right) HeLa cells were cotransfected with His-hnRNP K (lane 13) or its Pro-Ala variants (lanes 14 to 20) and with the constitutively active variant Src(KP) (lanes 13 to 20). (A) HeLa cell lysate was resolved by SDS-PAGE and analyzed in Western blot assays with antibodies against the His tag [His-ab], Src [Src-ab], Src phosphorylated at Y416 [Src(pY416)-ab], and GAPDH [GAPDH-ab] as a loading control. (B) His-tagged hnRNP K or its variants were immunoprecipitated with an anti-His antibody, resolved by SDS-PAGE, and analyzed by Western blot assays with antibodies against the His tag (His-ab), p-Tyr (pY-ab), and c-Src (Src-ab). wt, wild type.
FIG. 5.
FIG. 5.
The proline motifs P2 and P3 are sufficient to activate c-Src in vitro. Src(wt) (lanes 1 to 6) was immunopurified from transfected HeLa cells. Equal amounts, as shown in a Western blot assay with a Src-ab, were incubated in the presence of ATP with equal amounts of recombinant His-hnRNP K (lane 2) or its variant His-hnRNP K(P2[2,3]P3[1,2]) (lanes 3 to 6), as shown in a Western blot assay with an anti-His antibody (His-ab) and increasing amounts of the peptide representing P2 and P3 (aa 285 to 318) (lanes 4 to 6). Phosphorylation of His-hnRNP K or its variant was analyzed in a Western blot assay with a pY-ab.
FIG. 6.
FIG. 6.
hnRNP K activates c-Src but not wild-type Lyn, Fyn, or Lck. HeLa cells were transiently transfected with c-Src (lanes 1 to 3), Lyn (lanes 5 to 7), Fyn (lanes 9 to 11), or Lck (lanes 13 to 15) and with His-hnRNP K (lanes 2, 6, 10, and 14) or His-hnRNP K(P2[2,3]P3[1,2]) (lanes 3, 7, 11, and 15) or no cDNAs (lanes 4, 8, 12, and 16). (A) HeLa cell lysate was resolved by SDS-PAGE and analyzed in Western blot assays using antibodies against the His tag (His-ab), against Src, Lyn, Fyn, or Lck (kinase-ab), against p-Tyr (pY-ab), and against GAPDH (GAPDH-ab) as a loading control. (B) His-tagged hnRNP K or its variant was immunoprecipitated with an anti-His antibody, resolved by SDS-PAGE, and analyzed by Western blot assays using antibodies against the His tag (His-ab), against Src, Lyn, Fyn, or Lck (kinase-ab), and against p-Tyr (pY-ab). The Western blots for Lyn and Fyn and p-Tyr had to be overexposed to detect a signal.
FIG. 7.
FIG. 7.
Summary of the results of the in vitro protein-protein interaction assays. GST-pull down experiments with recombinant GST-hnRNP K or deletion variants representing the peptides indicated on the left. c-Src was synthesized in the presence of [35S]Met in a coupled transcription-translation assay in rabbit reticulocyte lysate. Equal amounts of 35S-labeled c-Src were incubated with GST-tagged hnRNP K or its deletion variants coupled to glutathione-Sepharose. The bound c-Src was released from the GST-tagged proteins under denaturing conditions and analyzed by SDS-PAGE and autoradiography. The binding (bdg.) of 35S-labeled c-Src to the GST-tagged hnRNP K or its deletion variants is indicated on the left.
FIG. 8.
FIG. 8.
Visualization of the interaction between hnRNP K and c-Src in living cells using BiFC analysis. (A) HeLa cells were cotransfected with cDNAs coding for V1-Src, V2-hnRNP K singly or together, V1-Src(Y416F), and V2-hnRNP K, V1-Src, and V2-hnRNP K(P2[2,3]P3[1,2]) as indicated. (Β) HeLa cells were cotransfected with V1-Src together with V2-hnRNP K(1-218), V2-hnRNP K(1-242), V2-hnRNP K(Δ216-226), V2-hnRNP K{(Δ216-226),(P2[2,3]P3[1,2])}, V2-hnRNP (K-E1-K), and V2-hnRNP {(K-E1-K)(P2[2,3]P3[1,2])} as indicated. For panels A and B, the expression of c-Src or Src(Y416F) and hnRNP K or its variants was determined by specific antibodies and Cy3 (c-Src)- or Cy5 (hnRNP K)-labeled secondary antibodies. The Venus BiFC analysis visualized direct protein-protein interaction. DAPI, 4′,6′-diamidino-2-phenylindole.
FIG. 8.
FIG. 8.
Visualization of the interaction between hnRNP K and c-Src in living cells using BiFC analysis. (A) HeLa cells were cotransfected with cDNAs coding for V1-Src, V2-hnRNP K singly or together, V1-Src(Y416F), and V2-hnRNP K, V1-Src, and V2-hnRNP K(P2[2,3]P3[1,2]) as indicated. (Β) HeLa cells were cotransfected with V1-Src together with V2-hnRNP K(1-218), V2-hnRNP K(1-242), V2-hnRNP K(Δ216-226), V2-hnRNP K{(Δ216-226),(P2[2,3]P3[1,2])}, V2-hnRNP (K-E1-K), and V2-hnRNP {(K-E1-K)(P2[2,3]P3[1,2])} as indicated. For panels A and B, the expression of c-Src or Src(Y416F) and hnRNP K or its variants was determined by specific antibodies and Cy3 (c-Src)- or Cy5 (hnRNP K)-labeled secondary antibodies. The Venus BiFC analysis visualized direct protein-protein interaction. DAPI, 4′,6′-diamidino-2-phenylindole.
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