Isoform-specific contribution of protein kinase C to prion processing.

The cellular prion protein (PrP(c)) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrP(c). First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCalpha lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCalpha-, PKCdelta- and PKCepsilon-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCepsilon-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCalpha, delta and epsilon, but not zeta, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production.

Inhibition of PKCepsilon improves glucose-stimulated insulin secretion and reduces insulin clearance.

In type 2 diabetes, pancreatic beta cells fail to secrete sufficient insulin to overcome peripheral insulin resistance. Intracellular lipid accumulation contributes to beta cell failure through poorly defined mechanisms. Here we report a role for the lipid-regulated protein kinase C isoform PKCepsilon in beta cell dysfunction. Deletion of PKCepsilon augmented insulin secretion and prevented glucose intolerance in fat-fed mice. Importantly, a PKCepsilon-inhibitory peptide improved insulin availability and glucose tolerance in db/db mice with preexisting diabetes. Functional ablation of PKCepsilon selectively enhanced insulin release ex vivo from diabetic or lipid-pretreated islets and optimized the glucose-regulated lipid partitioning that amplifies the secretory response. Independently, PKCepsilon deletion also augmented insulin availability by reducing both whole-body insulin clearance and insulin uptake by hepatocytes. Our findings implicate PKCepsilon in the etiology of beta cell dysfunction and highlight that enhancement of insulin availability, through separate effects on liver and beta cells, provides a rationale for inhibiting PKCepsilon to treat type 2 diabetes.

Defective IgG2a/2b class switching in PKC alpha-/- mice.

Using model tumor T cell lines, protein kinase C (PKC) alpha has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKCalpha null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3(+) PKCalpha-deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF-kappaB, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN-gamma production are severely impaired in PKCalpha(-/-) CD3(+) T cells. Consistently, PKCalpha-deficient CD3(+) T cells from OVA-immunized PKCalpha-deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKCalpha-deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKCalpha(-/-) B cell proliferation are unimpaired. Our genetic data, thus, define PKCalpha as the physiological and nonredundant PKC isotype in signaling pathways that are necessary for T cell-dependent IFN-gamma production and IgG2a/2b Ab responses.

Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport.

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.

B cell progenitors are arrested in maturation but have intact VDJ recombination in the absence of Ig-alpha and Ig-beta.

Ig-alpha and Ig-beta mediate surface expression and signaling of diverse B cell receptor complexes on precursor, immature, and mature B cells. Their expression begins before that of the Ig chains in early progenitor B cells. In this study, we describe the generation of Ig-alpha-deficient mice and their comparative analysis to mice deficient for Ig-beta, the membrane-IgM, and recombination-activating gene 2 to determine the requirement of Ig-alpha and Ig-beta in survival and differentiation of pro-B cells. We find that in the absence of Ig-alpha, B cell development does not progress beyond the progenitor stage, similar to what is observed in humans lacking this molecule. However, neither in Ig-alpha- nor in Ig-beta-deficient mice are pro-B cells impaired in V(D)J recombination, in the expression of intracellular Ig micro-chains, or in surviving in the bone marrow microenvironment. Finally, Ig-alpha and Ig-beta are not redundant in their putative function, as pro-B cells from Ig-alpha and Ig-beta double-deficient mice are similar to those from single-deficient animals in every aspect analyzed.