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RNA-dependent phosphorylation of a nuclear RNA binding protein - PubMed

  • ️Wed Jan 01 1997

RNA-dependent phosphorylation of a nuclear RNA binding protein

P A Fung et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The human C1 heterogeneous nuclear ribonucleoprotein particle protein (hnRNP protein) undergoes a cycle of phosphorylation-dephosphorylation in HeLa cell nuclear extracts that modulates the binding of this protein to pre-mRNA. We now report that hyperphosphorylation of the C1 hnRNP protein is mediated by a kinase activity in nuclear extracts that is RNA-dependent. Although the basal phosphorylation of the C1 hnRNP protein in nuclear extracts reflects a casein kinase II-type activity, its RNA-dependent hyperphosphorylation appears to be mediated by a different kinase. This is indicated by the unresponsiveness of the RNA-stimulated hyperphosphorylation to casein kinase II inhibitors, and the distinct glycerol gradient sedimentation profiles of the basal versus RNA-stimulated C1 hnRNP protein phosphorylation activities from nuclear extracts. RNA-dependent phosphorylation was observed both for a histidine-tagged recombinant human C1 hnRNP protein added to nuclear extracts and also for the endogenous C1 hnRNP protein. Additional results rule out protein kinase A, protein kinase C, calmodulin-dependent protein kinase II, and double-stranded RNA-activated protein kinase as the enzymes responsible for the RNA-dependent hyperphosphorylation of the C1 hnRNP protein. These results reveal the existence in nuclear extracts of an RNA-dependent protein kinase activity that hyperphosphorylates a known pre-mRNA binding protein, and define an additional element to be integrated into the current picture of how nuclear proteins are regulated by phosphorylation.

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Figures

Figure 1
Figure 1

Nuclear RNA stimulates C1 hnRNP protein phosphorylation. C1 hnRNP protein was incubated in nuclear extracts with [γ-32P]ATP, as detailed in Materials and Methods, and other additions as indicated. Lanes: 1, control (no other additions); 2, 1 μM okadaic acid; 3, 150 μg/ml nuclear extract RNA (no okadaic acid); 4, 150 μg/ml nuclear extract RNA plus 6 mM 2,3-bisphosphoglycerate; 5, 150 μg/μl nuclear extract RNA plus 12 mM 2,3-bisphosphoglycerate; 6, control (no other additions); 7, 50 μg/ml nuclear extract RNA; 8, 100 μg/ml nuclear extract RNA; 9, 200 μg/ml nuclear extract RNA; 10, C1 hnRNP protein incubated in micrococcal nuclease treated Jurkat nuclear extract as in lane 1, no added RNA; and 11, C1 hnRNP protein incubated as in lane 10 but with HeLa nuclear extract RNA, 75 μg/ml. Samples were subjected to gel electrophoresis in 14% polyacrylamide gels as described.

Figure 3
Figure 3

RNA also elicits hyperphosphorylation of endogenous C1 hnRNP protein in nuclear extract. HeLa nuclear extract (not pretreated with micrococcal nuclease) was incubated for 30 min (at 30°C) in the presence or absence of added nuclear extract RNA (final concentration of added RNA, 75 μg/ml) and then for an additional 60 min (at 30°C) with [γ-32P]GTP. The reactions were then processed as described followed by incubation with protein A-Sepharose coupled 4F4 monoclonal antibody for 2 hr at 4°C. After washing the resin, the bound C1 hnRNP protein was eluted (4, 5, 9, 10) and analyzed by electrophoresis and autoradiography. Lanes: 1, no added RNA; 2, added nuclear extract RNA.

Figure 2
Figure 2

RNA stimulation of C1 hnRNP protein phosphorylation does not require prior micrococcal nuclease treatment of the nuclear extract. C1 hnRNP protein was incubated with [γ-32P]ATP as in Fig. 1, except that the HeLa nuclear extract was not pretreated with micrococcal nuclease. Lanes: 1, control (no other additions); 2, 1 μM okadaic acid; and 3, 150 μg/ml nuclear extract RNA (no okadaic acid).

Figure 4
Figure 4

CKII-mediated phosphorylation of C1 hnRNP protein is not stimulated by RNA. C1 hnRNP protein was incubated for 2 hr at 30°C in the same buffer as used in Fig. 1 (but no nuclear extract) in the presence or absence of human CKII (Boehringer Mannheim) at a final concentration of 15 milliunits/ml, and in the presence or absence of HeLa nuclear extract RNA, at a final concentration of 75 μg/ml. Lanes: 1, C1 hnRNP protein and purified CKII; 2, C1 hnRNP protein, no CKII; 3, C1 hnRNP protein and CKII plus nuclear extract RNA; 4, C1 hnRNP protein plus nuclear extract RNA. Results similar to those shown in the figure were obtained when the incubation period of C1 hnRNP protein with CKII was 30 min or 1 hr. In addition to showing that the phosphorylation of C1 hnRNP protein by CKII is not stimulated by RNA (lanes 1 vs. 3), this experiment also indicates that the C1 hnRNP protein does not undergo autophosphorylation under these experimental conditions, either in the presence or absence of RNA (lanes 2 and 4).

Figure 5
Figure 5

Glycerol gradient centrifugation resolves the RNA-stimulated kinase activity from the basal C1 hnRNP protein phosphorylation activity. Micrococcal nuclease-treated HeLa nuclear extract was layered on 10–30% (vol/vol) glycerol gradients and centrifuged for 18 hr at 40,000 rpm (Beckman SW41 rotor) at 4°C. The gradients were fractionated, and each fraction was divided into two equal portions. One was assayed for C1 hnRNP protein phosphorylation without exogenous RNA, and the other was assayed for C1 hnRNP protein phosphorylation in the presence of added nuclear RNA (final concentration, 125 μg/ml). The proteins were displayed by electrophoresis and autoradiograms exposed in the linear range of the film’s dpm vs. silver grain exposure curve were subjected to quantitative densitometry. The amounts of 32P in the C1 hnRNP protein bands were summed and plotted as a function of gradient position. •, No added RNA; ○, with nuclear RNA. The S values indicated by the arrows were estimated by interpolation of the gradient positions of apoferritin (18S) and alcohol dehydrogenase (8S) standards (18).

Figure 6
Figure 6

Mechanistic possibilities for RNA-stimulation of C1 hnRNP protein hyperphosphorylation. In all three cases, the reaction pathway starts with the C1 hnRNP protein in its basal state of phosphorylation, indicated schematically by the presence of a single phosphoamino acid. (In reality, the basal state of C1 hnRNP protein phosphorylation in HeLa nuclear extracts consists of two phosphorylated serine residues, viz. Ser-107 and Ser-247; P. Dwen and T.P., unpublished results.) (I) The stimulating RNA (or an RNA-protein complex assembled from it, designated “RNP”) binds to the C1 hnRNP protein and changes its conformation into one conducive to hyperphosphorylation. (II) The effect of the RNA (or RNP) is to activate a latent kinase (KI). (III) The RNA itself mobilizes the labile energy in the β-γ phosphodiester bond of ATP (25) to catalyze, as a ribozyme, phosphate group transfer to the C1 hnRNP protein. Although III is very speculative, an ATP-hydrolytic ribozyme, viz. polynucleotide kinase, has been identified in RNA sequence space (26) and other ATP-binding RNAs have been observed, as have RNAs that act catalytically on amino acids (e.g., ref. 27).

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