ABSTRACT
This study explores the links between the GTPase RhoA and the serine kinase protein kinase D (PKD) during thymocyte development. The rationale is that RhoA and PKD regulate common biological responses during T cell development, but there is nothing known about their interdependence. In fibroblasts, Rho function is required for activation of PKD catalytic activity. However, the data show that activation of Rho is neither sufficient nor essential for PKD activation in T cells. One alternative explanation for the apparent convergence of PKD and Rho signaling in T cells is that PKD responses might be Rho-dependent. To address this latter possibility, we probed the Rho requirements for the actions of constitutively active PKD mutants in pre-T cells of transgenic mice. Active PKD can localize to either the plasma membrane or the cytosol, and we therefore compared the Rho requirements for the actions of membrane- or cytosol-localized PKD. Here we show that membrane-localized PKD regulation of pre-T cell differentiation is Rho-dependent, but the actions of cytosol-localized PKD are not. These studies demonstrate that a Rho requirement for PKD activation is not ubiquitous. Moreover, links between PKD and Rho are determined by the cellular location of PKD.
Subject(s)
Protein Kinase C/chemistry , rhoA GTP-Binding Protein/chemistry , rhoA GTP-Binding Protein/physiology , ADP Ribose Transferases/metabolism , Animals , Botulinum Toxins/metabolism , Cell Membrane/metabolism , Clostridium botulinum/metabolism , Cytosol/metabolism , Fibroblasts/metabolism , Humans , Jurkat Cells , Mice , Mice, Transgenic , Protein Kinase C/physiology , T-Lymphocytes/metabolismABSTRACT
The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.
Subject(s)
Diglycerides/metabolism , Lymphocytes/metabolism , Protein Kinase C/metabolism , Type C Phospholipases/metabolism , Amino Acid Motifs , Animals , Cell Line, Tumor , Chickens , Enzyme Activation , Humans , Lymphocytes/enzymology , Mice , Mutation/genetics , Phospholipase C gamma , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/chemistry , Protein Kinase C/genetics , Protein Structure, Tertiary , Serine/metabolism , Type C Phospholipases/deficiency , Type C Phospholipases/geneticsABSTRACT
Protein kinase D (PKD) is an antigen receptor-activated serine kinase localized at either the plasma membrane or the cytosol of lymphocytes. To probe PKD function at these different locations, transgenesis was used to target active PKD either to the membrane or cytosol of pre-T cells. In recombinase gene null pre-T cells, membrane and cytosolic active PKD both induced differentiation reminiscent of beta selection: downregulation of CD25 and upregulation of CD2 and CD5. Active PKDs also induced pre-T cell proliferation, although this response was not universal to all thymocyte subsets. There were two striking differences between the actions of the differentially localized PKDs. Membrane but not cytosolic PKD could induce expression of CD8 and CD4 in recombinase null mice; cytosolic but not membrane PKD suppressed Vbeta to DJbeta rearrangements of the TCRbeta chain locus in wild-type T cells. PKD function is thus determined by its intracellular location and cell context.
Subject(s)
Protein Kinase C/metabolism , Animals , Cell Membrane/metabolism , Cytosol/metabolism , Mice , Mutation , Protein Kinase C/genetics , Receptors, Antigen, T-Cell/metabolismABSTRACT
The Neuropeptide Y (NPY) family of neuropeptides exert their function through a family of heptahelical G-protein coupled receptors regulating essential physiological processes. A 97 base pair intron (intron IV) intervenes the coding sequence of the human NPY Y1 receptor (hY1) gene and was found frequently retained at variable levels in poly A+ mRNA isolated from multiple human tissues. When included in hY1 expression vectors, either in its natural position or 5' of the hY1 cDNA, intron IV mediated a significant increase of both hY1 mRNA and corresponding functional receptor protein in transfected mammalian cells, implying an in vivo regulatory function of the endogenous intron. Our results further indicate that the nuclear history of the hY1 pre-mRNA influence ectopic hY1 production through post-transcriptional mechanisms and argues against intron IV acting as a transcriptional enhancer as well as the possibility that a putative hY1 related 5TM accessory protein encoded by the non-spliced hY1 mRNA would facilitate hY1 production on a post-translational level.
