We show here for the first time that a signal transduction pathway can control alternative splice site selection in vivo by regulating the cellular localization of pre-mRNA splicing factors. We have shown that the bidirectional transport of hnRNP A1 between the nucleus and the cytoplasm is not a constitutive process. Instead, it is subject to modulation by extracellular signals, which trigger rapid changes in the distribution of the protein between these cellular compartments. The relocalization of hnRNP A1 to the cytoplasm in stress-activated cells is mediated by the MKK3/6
-p38 pathway, which activation is both required and sufficient for hnRNP A1 cytoplasmic accumulation. At the same time, OSM leads to phosphorylation of hnRNP A1 with kinetics that parallel the localization of hnRNP A1. Although the role of protein phosphorylation in splicing is unclear, recent observations suggest that phosphorylation modulates protein–protein interactions within the spliceosome and controls the subnuclear distribution of SR proteins in interphase and mitosis (Misteli 1999
). In addition, phosphorylation of splicing regulators may be a way of regulating the subcellular distribution of antagonistic factors, and the resulting changes in the ratios of these factors in the nucleus may in turn affect splicing activity. This proposed mechanism represents a novel way of regulating alternative splice-site selection.
hnRNP A1 is directly phosphorylated in vitro by PKA, casein kinase II (CKII) and ζPKC (Cobianchi et al. 1993
; Municio et al. 1995
), but is not a direct target of p38 kinase (not shown). Phosphorylation by PKA significantly alters the properties of hnRNP A1, suppressing its strand annealing ability with no effect on its nucleic acid binding capacity (Cobianchi et al. 1993
). We have shown that stress signals induced the cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. Although the expression of a permanently active mutant of ζPKC promotes the cytoplasmic accumulation of hnRNP A1 in proliferating cells (Municio et al. 1995
), transfection of a kinase-inactive dominant-negative mutant of ζPKC had no effect on the cytoplasmic accumulation of hnRNP A1 in response to stress (not shown). Regarding the potential role of PKA in this pathway, exposure of cells to dibutyryl cAMP or cholera toxin, which potently activate PKA in vivo, did not induce the cytoplasmic accumulation of hnRNP A1 (not shown). Therefore, neither ζPKC nor PKA kinases appear to play a role during the stress-induced subcellular redistribution of hnRNP A1.
The mechanism whereby p38 kinase regulates hnRNP A1 cytoplasmic accumulation is presently unknown. The large increase in phosphorylation of hnRNP A1 upon osmotic shock may be part of the signal transduction pathway that leads to its altered partitioning between the nucleus and the cytoplasm. Alternatively, phosphorylation of hnRNP A1 may be a consequence of its accumulation and the cytoplasm, where it may be exposed to a kinase with which it does not normally interact. Although hnRNP A1 it is not a direct substrate for p38, it is known that p38 kinase activates other kinases, such as MAPKAP kinase-2 and -3 (McLaughlin et al. 1996
), which in turn may directly phosphorylate hnRNP A1, altering its shuttling properties. MAPKAP kinase-2 and p38 form a complex in the nucleus of unstimulated cells, where p38 kinase is inactive. In stimulated cells, phosphorylation of MAPKAP kinase-2 by p38 leads to a conformational change in MAPKAP kinase-2, which results in the masking of a nuclear localization signal (and perhaps the exposure of a nuclear export signal) that causes the relocalization of the p38-MAPKAP kinase-2 complex to the cytoplasm. Thus, MAPKAP kinase-2 acts both as an effector of p38 by phosphorylating substrates and also as a determinant of the cellular localization of p38 (Ben-Levy et al. 1998
; Engel et al. 1998
). This cytoplasmic translocation event may allow p38 and MAPKAP kinase-2 to phosphorylate cytoplasmic targets.
Alternatively, the effect of p38 on hnRNP A1 cytoplasmic accumulation could be indirect, through modulation of the activity of proteins involved in the nucleo-cytoplasmic transport of hnRNP A1. In this regard, a novel receptor-mediated nuclear import pathway, which is distinct from the classical importin-mediated NLS pathway, has been described. This new pathway involves a novel 90-kD protein, transportin, which physically interacts with, and serves to facilitate the nuclear import of, hnRNP A1 (Pollard et al. 1996
). An attractive possibility is that the p38 kinase cascade, through the phosphorylation of hnRNP A1, modulates its interaction with transportin, resulting in a decreased rate of nuclear import. However, it is not clear whether the altered subcellular distribution of hnRNP A1 following stress signaling is the result of inhibition of nuclear import or of an increased rate of nuclear export.
For the majority of nuclear events regulated by extracellular stimuli, the targets of the signaling pathways are transcription factors. However, other nuclear activities, such as mRNA processing, are also potential targets for these signaling pathways. One example of a growth factor–regulated splicing event is the processing of the PTP-1B pre-mRNA, which gives rise to a unique mRNA isoform via alternative splicing upon stimulation of quiescent cells with a variety of growth factors (Shiffrin and Neel 1993
). The nuclear cap-binding complex (CBC) has also been identified as a target for growth factor receptor-coupled signal transduction (Wilson et al. 1999
). The ability of growth factors to stimulate the capped RNA-binding activity correlates with growth factor stimulation of splicing activity. The CBC receives inputs from multiple pathways, since it is activated by the Ras-Raf-MEK pathway and also by stress-activated signaling pathways. However, the subcellular localization of both CBC protein subunits (CBP80 and CBP20) is not affected by growth factor stimulation (Wilson et al. 1999
The results reported here establish a new link between signaling cascades that are central to the control of important cell functions, such as the stress response, and the control of gene expression by mRNA processing. We showed that stress signals induce the cytoplasmic accumulation of hnRNP A1, concomitantly with changes in the alternative splicing of a cotransfected reporter. The cytoplasmic accumulation of hnRNP A1 after OSM presumably causes an altered ratio of the antagonistic alternative splicing factors SF2/ASF and hnRNP A1 in the cell nucleus. Indeed, the alternative splicing pattern of the E1A reporter mini-gene shows a decrease of the relative level of 9S transcripts in cells exposed to stress signals compared with untreated cells, as would be expected for splicing in the presence of reduced levels of hnRNP A1. In addition to the effects attributable to hnRNP A1, it is possible that other splicing factors are also altered by osmotic stress and p38 activation, contributing to the overall effect on alternative splicing. For example, we have shown that hnRNP B1 also accumulates in the cytoplasm under these conditions (), and this protein is structurally and functionally closely related to hnRNP A1 (Mayeda et al. 1994
The present findings demonstrate that changes mediated by signaling can modulate alternative splicing by altering the localization of known splicing regulators. This regulatory mechanism may turn out to be a general one, by which different signaling pathways may affect the nuclear ratio of specific antagonistic splicing regulators, thereby modulating alternative splicing regulation of particular pre-mRNAs.