The human CD45 gene encodes a protein-tyrosine phosphatase that exhibits differential isoform expression in resting and activated T cells due to alternative splicing of three variable exons. Previously, we have used biochemical methods to identify two regulatory proteins, hnRNP L and PSF, which contribute to the activation-induced skipping of CD45 via the ESS1 regulatory element in variable exon 4. Here we report the identification of a third CD45 regulatory factor, hnRNP L-like (hnRNP LL), via a cell-based screen for clonal variants that exhibit an activation-like phenotype of CD45 splicing even under resting conditions. Microarray analysis of two splicing-altered clones revealed increased expression of hnRNP LL relative to wild-type cells. We further demonstrate that both the expression of hnRNP LL protein and its binding to ESS1 are up-regulated in wild-type cells upon activation. Forced overexpression of hnRNP LL in wild-type cells results in an increase in exon repression, while knockdown of hnRNP LL eliminates activation-induced exon skipping. Interestingly, analysis of the binding of hnRNP L and hnRNP LL to mutants of ESS1 reveals that these proteins have overlapping, but distinct binding requirements. Together, these data establish that hnRNP LL plays a critical and unique role in the signal-induced regulation of CD45 and demonstrate the utility of cell-based screens for the identification of novel splicing regulatory factors.

Cells can regulate their protein repertoire in response to extracellular stimuli via alternative splicing; however, the mechanisms controlling this process are poorly understood. The CD45 gene undergoes alternative splicing in response to T-cell activation to regulate T-cell function. The ESS1 splicing silencer in CD45 exon 4 confers basal exon skipping in resting T cells through the activity of hnRNP L and confers activation-induced exon skipping in T cells via previously unknown mechanisms. Here we have developed an in vitro splicing assay that recapitulates the signal-induced alternative splicing of CD45 and demonstrate that cellular stimulation leads to two changes to the ESS1-bound splicing regulatory complex. Activation-induced posttranslational modification of hnRNP L correlates with a modest increase in the protein's repressive activity. More importantly, the splicing factor PSF is recruited to the ESS1 complex in an activation-dependent manner and accounts for the majority of the signal-regulated ESS1 activity. The associations of hnRNP L and PSF with the ESS1 complex are largely independent of each other, but together these proteins account for the total signal-regulated change in CD45 splicing observed in vitro and in vivo. Such a combinatorial effect on splicing allows for precise regulation of signal-induced alternative splicing.To maintain viability most, if not all, cells within an organism must be capable of responding to a changing environment. For example, neuronal and muscle cells must respond to activation to promote behaviors and movement, while lymphoid cells must respond to an immune challenge to prevent or control infection. Such flexibility requires that individual cells have the ability to change function rapidly and precisely in response to a given stimulus. In general, cellular responsiveness is accomplished through the activity of signal transduction cascades that ultimately alter protein expression or function via numerous mechanisms. Much of the previous work on signal-regulated protein expression has focused on the regulation of transcription; however, there is growing recognition of the abundance and importance of signal-induced changes in alternative splicing (2, 11, 13).Alternative splicing is the process by which exons of a given gene are differentially included or excluded from the final message, such that a single gene can be processed into multiple discrete mRNAs (3, 16). Since each of these variant mRNAs typically encodes a unique protein of distinct function, changes in the splicing pattern of a gene can have a profound effect on protein expression and cellular activity. It has recently become apparent that numerous genes undergo changes in splicing patterns upon neuronal depolarization (2, 13) or T-cell activation (11), suggesting a broad role for this mode of gene regulation in determining the physiologic response of cells to stimulation. Thus, understanding the mechanisms by which extracellular stimuli can induce changes in the splicing regulatory machinery is of prima...

Ras GTPases are activated by RasGEFs and inactivated by RasGAPs, which stimulate the hydrolysis of RasGTP to inactive RasGDP. GTPase-impairing somatic mutations in RAS genes, such as KRASG12D, are among the most common oncogenic events in metastatic cancer. A different type of cancer Ras signal, driven by overexpression of the RasGEF RasGRP1 (Ras guanine nucleotide-releasing protein 1), was recently implicated in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and murine models, in which RasGRP1 T-ALLs expand in response to treatment with interleukins (ILs) 2, 7 and 9. Here, we demonstrate that IL-2/7/9 stimulation activates Erk and Akt pathways downstream of Ras in RasGRP1 T-ALL but not in normal thymocytes. In normal lymphocytes, RasGRP1 is recruited to the membrane by diacylglycerol (DAG) in a phospholipase C-γ (PLCγ)-dependent manner. Surprisingly, we find that leukemic RasGRP1-triggered Ras-Akt signals do not depend on acute activation of PLCγ to generate DAG but rely on baseline DAG levels instead. In agreement, using three distinct assays that measure different aspects of the RasGTP/GDP cycle, we established that overexpression of RasGRP1 in T-ALLs results in a constitutively high GTP-loading rate of Ras, which is constantly counterbalanced by hydrolysis of RasGTP. KRASG12D T-ALLs do not show constitutive GTP loading of Ras. Thus, we reveal an entirely novel type of leukemogenic Ras signals that is based on a RasGRP1-driven increased in flux through the RasGTP/GDP cycle, which is mechanistically very different from KRASG12D signals. Our studies highlight the dynamic balance between RasGEF and RasGAP in these T-ALLs and put forth a new model in which IL-2/7/9 decrease RasGAP activity.

The Ets-binding site within the basal transcription element (BTE) of the rat prolactin (rPRL) promoter is critical for both basal and growth factor-regulated rPRL gene expression. Here we report the purification and identification of the factor that binds to the BTE. This factor was purified from GH3 pituitary nuclear extracts using ammonium sulfate fractionation, heparin-Sepharose and Mono Q chromatography, and BTE-affinity magnetic beads. We purified two proteins of 57 and 47 kDa and identified the 57-kDa protein by mass spectrometry as the Ets factor GABP␣. Western blot analysis identified the 47-kDa protein as GABP␤1. Co-transfection of dominant-negative GABP␤1 blocks prolactin promoter basal activity by 85-88% in GH3 cells in the presence or absence of FGF-4. Additionally, expression of wild-type GABP␣/␤1 selectively activates a minimal BTE promoter 24 -28-fold in GH3 cells, and this activation is dependent on the Ets-binding site. Finally, small interfering RNA depletion of GABP in GH3 cells results in the loss of prolactin protein. Thus, we have identified GABP␣/GABP␤1 as a critical and functionally relevant Ets factor that regulates rPRL promoter activity via the BTE site.