Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein

Abstract

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleocytoplasmic transport of PTB is regulated by the 3,5-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB. T he heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins are involved in a variety of processes in mRNA metabolism including pre-mRNA splicing, mRNA transport, and translation (1). These processes are often regulated by cellular signaling pathways, but how this dynamic control is achieved is mostly unknown. Some hnRNP proteins are known to be phosphorylated, but in most cases the particular kinase that modifies the protein is not known. Moreover, it is generally not clear how such modifications affect protein function. HnRNP proteins localize primarily in the nucleus at steady state but some of them engage in nucleo-cytoplasmic shuttling (2, 3). There are examples of hnRNP localization being altered by specific signaling pathways (4, 5).The polypyrimidine tract-binding protein (PTB, or hnRNP I) has both nuclear and cytoplasmic functions. In the nucleus, it is a splicing repressor of a number of alternative exons (6, 7). In the cytoplasm, PTB plays a role in viral RNA translation through internal ribosome entry sites (8-11). Also in the cytoplasm, PTB is implicated in mRNA localization in Xenopus oocytes (12). The protein contains four RNA-recognition-motif type RNA binding domains (RRMs) and a conserved N-terminal domain. The N-terminal 55-aa segment of PTB contains both nuclear import and export signals and is sufficient to allow some nucleocytoplasmic shuttling in heterokaryon assays (13-17). There is an additional sequence within RRM2 that enhances nuclear export (17). At steady state, PTB is highly enriched in the nucleus, but its distribution must be regulated because the protein also has cytoplasmic functions. However, little is known about this regulation and what cellular signaling...

Cytoplasmic Relocalization of Heterogeneous Nuclear Ribonucleoprotein A1 Controls Translation Initiation of Specific mRNAs

“…hnRNP I (PTB) is an IRES trans-acting factor involved in translation regulation of many IRES-containing mRNAs (Valcarcel and Gebauer, 1997;Spriggs et al, 2005;Bushell et al, 2006). Interestingly, it was found that direct protein kinase A phosphorylation of PTB modulates its nucleocytoplasmic distribution (Xie et al, 2003). In all these cases, it remains to be determined whether the cytoplasmic relocalization directly controls cytoplasmic translation.…”

Control of alternative pre-mRNA splicing by Ca++ signals

“…Relocalization of splicing factors in response to Ca ++ signals has also been observed, as in the cases of other cell signals [167][168][169][170]. In cortical neurons, thapsigargin or ischemia treament induces Tra2 beta1 relocalization from the nucleus to the cytoplasm [99].…”

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

“…For example, it has been shown recently that nucleo-cytoplasmic shuttling of PTB-1 is regulated by the 3 0 -5 0 cyclic AMP-dependent protein kinase PKA. 67 Moreover, during apoptosis in certain cell types, PTB-1 has been shown to be cleaved by caspase 3 between RRM 1 and 2 resulting in relocation of this section of the protein to the cytoplasm, although the effect that this has on cellular IRES function is at present unknown. 68 PCBP1 is induced under cell stress conditions, mediated via signalling through the MAP kinase pathway, 69 a pathway which is known to be induced during apoptosis initiated by either anti-CD95, or TNFa.…”