Temporal differences in the dependency on phosphoinositide-dependent kinase 1 distinguish the development of invariant Valpha14 NKT cells and conventional T cells.

This study uses two independent genetic strategies to explore the requirement for phosphoinositide-dependent kinase-1 (PDK1) in the development of mature T cell populations from CD4/CD8 double-positive thymocytes. The data show that CD4/CD8 double-positive thymocytes that do not express PDK1 or express a catalytically inactive PDK1 mutant fail to produce mature invariant Vα14 NKT cells but can differentiate to conventional CD4, CD8, or regulatory T cell subsets in the thymus. The PDK1 requirement for Vα14 NKT cell development reflects that these cells require the PDK1 substrate protein kinase B to meet the metabolic demands for proliferative expansion in response to IL-15 or AgR stimulation. There is also constitutive PDK1 signaling in conventional α/ß T cells that is not required for lineage commitment of these cells but fine-tunes the expression of coreceptors and adhesion molecules. Also, although PDK1 is dispensable for thymic development of conventional α/ß T cells, peripheral cells are reduced substantially. This reflects a PDK1 requirement for lymphopenia-induced proliferation, a process necessary for initial population of the peripheral T cell niche in neonatal mice. PDK1 is thus indispensable for T cell developmental programs, but the timing of the PDK1 requirement is unique to different T cell subpopulations.

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D.

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.

Notch-induced T cell development requires phosphoinositide-dependent kinase 1.

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.

Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes.

In the present study, we have explored the impact of deleting a single allele of PDK1 in T cell progenitors on alpha/beta and gamma/delta T cell development. The data show that deleting a single allele of PDK1 allows differentiation of alpha/beta T cells but prevents their proliferative expansion in the thymus. Accordingly, mice with T cells that are haplo-insufficient for PDK1 have reduced numbers of thymocytes and alpha/beta peripheral T cells. T cell progenitors also give rise to gamma/delta T cells but in contrast to the loss of alpha/beta T cells in T-PDK1 null and haplo-insufficient mice, there were increased numbers of gamma/delta T cells. The production of alpha/beta T cells is dependent on the proliferative expansion of thymocytes and is determined by a balance between the frequency with which cells enter the proliferative phase of the cell cycle and rates of cell death. Herein, we show that PDK1 haplo-insufficient thymocytes have no defects in their ability to enter the cell cycle but show increased apoptosis. PDK1 thus plays a determining role in the development of alpha/beta T lymphocytes but does not limit gamma/delta T cell development.

Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1.

Bone morphogenetic proteins (BMPs) regulate multiple cellular processes, including cell differentiation and migration. Their signals are transduced by the kinase receptors BMPR-I and BMPR-II, leading to Smad transcription factor activation via BMPR-I. LIM kinase (LIMK) 1 is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin depolymerizing factor. During a search for LIMK1-interacting proteins, we isolated clones encompassing the tail region of BMPR-II. Although the BMPR-II tail is not involved in BMP signaling via Smad proteins, mutations truncating this domain are present in patients with primary pulmonary hypertension (PPH). Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4. A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1. This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.