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  1. Reviewer #3 (Public Review):

    This paper from He, Y. et al examines how PKC-theta in activated T cells controls RanBP2 nuclear pore subcomplex formation and nuclear translocation of NFkB, NFAT and AP1 family transcription factors. He, Y et al systematically pull apart a molecular mechanism showing that: 1) T cell receptor-activated PKC-theta localises to the nuclear envelope and associates with RanGAP1, 2) PKC-theta deficiency reduces nuclear localisation of import proteins and AP1-family transcription factors in mature mouse T cells and Jurkat cell line, but not primary mouse thymocytes 3) RanGAP1 is phosphorylated by PKC-theta and that phosphorylation of RanGAP1 on Ser504/Ser506 facilitates RanGAP1 sumoylation and is needed for association with other RanBP2 complex components and 4) that wildtype but not Ser504/506 mutant RanGAP1 can rescue nuclear translocation of transcription factors in RanGAP1 knockdown cells.

    A key strength of this work is that, for many key results, multiple methods for validating findings are used e.g. immunoblots of subcellular fractionation + confocal microscopy to show failure of c-Jun into the nucleus in Prkcq-/- mature T cells (Fig 3 G-H). Furthermore, although the majority of the molecular work takes advantage of the more tractable Jurkat cell line for dissection of molecular mechanism, a number of key points are validated in primary mouse or human T cells such as PKC-theta dependent TCR induced association of RanGAP1 with the nuclear pore (Fig 3D-E) and multiple methods of gene deletion were used e.g. siRNA, knockout mouse model and stable CRISPR deletion. The validation of a functionally meaningful phospho-site on the RanGAP1 protein is valuable for further understanding the biology of this protein.

    Immune receptor control of nuclear transport machinery has not been extensively studied but, as is highlighted by this study, is increasingly being understood as an important step in immune receptor control of transcription factor function. The molecular mechanism that is uncovered here is novel and interesting to the immunological community as it links TCR signalling to an indirect mechanism for regulating localisation of multiple key transcription factors for the T cell immune response.

    There are some concerns listed below. Addressing these concerns would add clarity to the manuscript and support some stated or implied conclusions.

    1. The data on the role of PKC-theta driven RanBP2 subcomplex translocation of AP1 transcription factors is largely limited to within 15 min of T cell activation. The broad statements of the paper e.g. line 427 - "PKC-theta plays an indispensable role in NPC assembly" imply that PKC-theta is essential for this process during long-term T cell receptor activation; however, whether PKC-theta deletion has long term impact on nuclear translocation after these first 15 minutes is not established. The demonstration that the RanGAP1 mutant is not able to induce IL-2 production over 24 hrs (Fig 6D) does support the model that a longer-term requirement for RanGAP1 phosphorylation on Ser504/506 is important for translocation and functional AP1 transcriptional outcomes in this system, but from the data presented it does not necessarily follow that PKC-theta is the only regulator of this beyond the 15 min of activation shown here. It is well established that AP1 transcription factors increase in expression for multiple hours after T cell activation and if PKC-theta deletion impact is not long lasting this could mean PKC-theta is important for the kinetics of AP1 translocation but not necessarily for final functional outcome after a longer period of stimulation as is implied here.

    2. It has been shown in the published literature the impact of PKC theta deletion on in vivo immune responses has been varied, with studies showing clearance of murine Listeria, LCMV, HSV. The manuscript currently lacks discussion around how the formation of a largely functional immune response in these contexts fits in with the strong defect in nuclear translocation of multiple important T cell transcription factors that they show here.

  2. Reviewer #2 (Public Review):

    PKC-theta is a critical signaling molecule downstream of T cell receptor (TCR), and required for T cell activation via regulating the activation of transcription factors including AP-1, NF-kB and NFAT. This manuscript revealed a novel function of PKC-theta in the regulation of the nuclear translocation of these transcription factors via nuclear pore complexes. This novel perspective for PKC-theta function advances our understanding T cell activation. The manuscript provided solid cellular and biochemical evidence to support the conclusions. However, nuclear pore complexes regulate the export and import essential components of cells, it is not clear whether PKC-theta selectively regulates the translocation of above transcription factors, or also other components, and whether regulates both import and export. It is essential to provide more substantial evidence to support the conclusion.

  3. Reviewer #1 (Public Review):

    The manuscript by He et al. reveals a novel role for PKC-theta, following T cell receptor (TCR) stimulation, in regulating the nuclear translocation of several key activation-dependent transcription factors by regulating the assembly of key components of the nuclear pore complex (NPC). The authors make use of T cell lines and primary T cells to show that following TCR stimulation, PKC-theta phosphorylates RanGAP1 to promote its interaction with Ubc9 and increase the sumoylation of RanGAP1, which, in turn, enhances assembly of the RanBP2 subcomplex of the NPC that then promotes the nuclear import of AP-1, NFAT and NFB. These conclusions are well supported by a rigorous experimental approach, which included the use of PKC-theta deficient, sumoyltion-defective, kinase-dead, and constitutively active mutants, and RanGAP1-deficient cells.

  4. Evaluation Summary:

    PKC-theta is known to regulate T cell activation, and this manuscript reveals a novel function of PKC-theta in the regulation of the nuclear pore complexes. The work by He and colleagues reveals that PKC-theta is recruited to the nuclear pore complex wherein it serves to regulate the assembly of key components of the RanBP2 subcomplex of the NPC, which in turn enables the translocation of AP1, NFkB and NFAT into the nucleus. However, these results need to be substantiated by additional experiments or by limiting the breath of the conclusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)