Sik (mouse Src-related intestinal kinase) and its orthologue BRK (human breast tumor kinase) are intracellular tyrosine kinases that are distantly related to the Src family and have a similar structure, but they lack the myristoylation signal. Here we demonstrate that Sik and BRK associate with the RNA binding protein Sam68 (Src associated during mitosis, 68 kDa). We found that Sik interacts with Sam68 through its SH3 and SH2 domains and that the proline-rich P3 region of Sam68 is required for Sik and BRK SH3 binding. In the transformed HT29 adenocarcinoma cell cell line, endogenous BRK and Sam68 colocalize in Sam68-SLM nuclear bodies (SNBs), while transfected Sik and Sam68 are localized diffusely in the nucleoplasm of nontransformed NMuMG mammary epithelial cells. Transfected Sik phosphorylates Sam68 in SNBs in HT29 cells and in the nucleoplasm of NMuMG cells. In functional studies, expression of Sik abolished the ability of Sam68 to bind RNA and act as a cellular Rev homologue. While Sam68 is a substrate for Src family kinases during mitosis, Sik/BRK is the first identified tyrosine kinase that can phosphorylate Sam68 and regulate its activity within the nucleus, where it resides during most of the cell cycle.

Human immunodeficiency virus (HIV) type 1 encodes an essential protein, Rev, which functions to transport unspliced and singly spliced viral transcripts from the nucleus to the cytoplasm to allow expression of the viral structural proteins. It has previously been reported that Sam68 synergistically stimulates Rev activity (T. Reddy et al., Nat. Med. 5:635-642, 1999). Here we report that the Sam68-like mammalian proteins SLM1 and SLM2 also stimulate Rev activity. Their stimulation ability cannot be attributed to a shuttling property, since Sam68, SLM1, and SLM2 do not display significant shuttling activity alone or in the presence of Rev. In addition, Sam68, SLM1, and SLM2 do not affect the equilibrium between unspliced and completely spliced HIV RNA. The C-terminally truncated Sam68 mutant (Sam68⌬C) previously observed to inhibit the Sam68-mediated stimulation of Rev activity (Reddy et al., 1999) also inhibits SLM1-and SLM2-mediated stimulation of Rev activity. This suggests that the mechanism by which Sam68, SLM1, and SLM2 stimulate Rev activity may be common. Sam68⌬C does not inhibit Rev activity by inhibiting Rev from shuttling between the nucleus and cytoplasm. Inhibition by Sam68⌬C is a consequence of its mislocalization to the cytoplasm, as evidenced by the fact that addition of an exogenous nuclear localization signal to Sam68⌬C restores nuclear localization and stimulation of Rev activity. We demonstrate that Sam68⌬C causes perinuclear accumulation of unspliced HIV env RNA and propose that Sam68⌬C inhibits Rev activity by sequestering Rev-responsive RNA away from the translation apparatus.Expression of the full complement of human immunodeficiency virus type 1 (HIV-1) proteins requires that incompletely spliced viral transcripts be transported from the nucleus to the cytoplasm for translation into the structural proteins of the virus (10,12,19,28). Nuclear export of the incompletely spliced transcripts is achieved through the action of an essential HIV accessory protein, Rev (11,43). Expressed from fully spliced viral RNA, Rev localizes to the nucleus through the interaction of its nuclear localization signal (NLS) (5,21,27,35) with the import receptor importin ␤ (49). Once in the nucleus, Rev interacts with the incompletely spliced viral transcripts. This interaction is mediated by a 240-nucleotide sequence, termed the Rev responsive element (RRE), present within the terminal intron of the incompletely spliced and unspliced viral transcripts (3,18,20,28,40,56). Nuclear export of the Rev-RNA complex is then mediated by interaction of the Rev nuclear export signal (NES) (13, 31) with the nuclear export receptor CRM1 (14, 45). Translation of the unspliced and singly spliced viral transcripts results in production of the structural proteins, and some accessory proteins of the virus (6). Rev thus acts as a regulator of the switch from early to late viral gene expression (9,25).It has recently been reported that Sam68, the 68-kDa Srcassociated substrate during mitosis (15,47), is a functional homologue of Rev...