Heterogenous nuclear ribonucleoprotein (hnRNP) A1 is an alternative splicing factor that is mainly nuclear, although it shuttles rapidly between nuclear and cytoplasmic compartments. Cells stressed by osmotic shock (OSM) activate the mitogen-activated protein kinase kinase3/6-p38 signaling pathway, which in turn results in accumulation of hnRNP A1 in the cytoplasm. This effect modulates alternative splicing regulation in vivo and correlates with increased hnRNP A1 phosphorylation. We have characterized the molecular mechanism involved in the cytoplasmic accumulation of hnRNP A1 in NIH 3T3 cells subjected to OSM. This treatment results in serine-specific phosphorylation within a C-terminal peptide, dubbed the ''F-peptide,'' which is adjacent to the M9 motif that mediates bidirectional transport of hnRNP A1. Analysis of mutants in which the F-peptide serines were replaced by aspartic acids or alanines showed that F-peptide phosphorylation is required for the subcellular redistribution of hnRNP A1 in cells subjected to OSM. Furthermore, F-peptide phosphorylation modulates the interaction of hnRNP A1 with transportin Trn1. Our findings suggest that the phosphorylation of F-peptide by cellsignaling pathways regulates the rate of hnRNP A1 nuclear import.shuttling ͉ alternative splicing ͉ stress signaling ͉ transportin ͉ p38 kinase H eterogenous nuclear ribonucleoprotein (hnRNP) plays an important role in all of the steps of mRNA metabolism (1, 2). The human hnRNP family consists of at least 24 members, which are among the most abundant nuclear proteins (3, 4). hnRNP A1, a member of the hnRNP A͞B subfamily, has been studied extensively and participates in the regulation of transcription, splicing, and mRNA export.hnRNP A1 binds RNA through two RNA recognition motif modules at its N terminus (amino acids . The C-terminal domain (amino acids 197-320) comprises several RGG repeats, which also contribute to RNA binding. The C terminus also includes a 38-aa sequence, the M9 motif (amino acids 268-305), that is involved in hnRNP A1 nuclear import and export (5-8). Although at steady state hnRNP A1 is predominantly nuclear, it shuttles rapidly between the nucleus and cytoplasm (9). The shuttling of hnRNP A1 is subject to regulation and is thought to play a role in cell proliferation, survival, and differentiation of normal and transformed cells (10). The shuttling of hnRNP A1 also is required for normal myelopoiesis and BCR͞ABL leukemogenesis (10).The molecular mechanism that regulates the nuclear export of hnRNP A1 is unknown. In contrast, the mechanism of hnRNP A1 nuclear import is better understood. Two transport receptors of the karyopherin-␤ family, Trn1 and Trn2b, interact directly with the M9 sequence and mediate hnRNP A1 import (11-13). hnRNP A1 transport requires an intact M9 motif, and a single amino acid change, G274A, abolishes both import and export of the protein (14).The subcellular distribution of hnRNP A1 is the result of steady-state control of its transport between the nucleus and the cytoplasm. However, it...