Protein kinase C delta (PKC delta) mediates apoptosis downstream of many apoptotic stimuli. Because of its ubiquitous expression, tight regulation of the proapoptotic function of PKC delta is critical for cell survival. Full-length PKC delta is found in all cells, whereas the catalytic fragment of PKC delta, generated by caspase cleavage, is only present in cells undergoing apoptosis. Here we show that full-length PKC delta transiently accumulates in the nucleus in response to etoposide and that nuclear translocation precedes caspase cleavage of PKC delta. Nuclear PKC delta is either cleaved by caspase 3, resulting in accumulation of the catalytic fragment in the nucleus, or rapidly exported by a Crm1-sensitive pathway, thereby assuring that sustained nuclear accumulation of PKC delta is coupled to caspase activation. Nuclear accumulation of PKC delta is necessary for caspase cleavage, as mutants of PKC delta that do not translocate to the nucleus are not cleaved. However, caspase cleavage of PKC delta per se is not required for apoptosis, as an uncleavable form of PKC delta induces apoptosis when retained in the nucleus by the addition of an SV-40 nuclear localization signal. Finally, we show that kinase negative full-length PKC delta does not translocate to the nucleus in apoptotic cells but instead inhibits apoptosis by blocking nuclear import of endogenous PKC delta. These studies demonstrate that generation of the PKC delta catalytic fragment is a critical step for commitment to apoptosis and that nuclear import and export of PKC delta plays a key role in regulating the survival/death pathway.

Oncogenic activation of KRAS occurs commonly in non-small cell lung cancer (NSCLC), but strategies to therapeutically target this pathway have been challenging to develop. Information about downstream effectors of KRAS remains incomplete and tractable targets are yet to be defined. In this study we investigated the role of Protein Kinase C delta (PKCδ) in KRAS dependent lung tumorigenesis using a mouse carcinogen model and human NSCLC cells. The incidence of urethane-induced lung tumors was decreased by 69% in PKCδ deficient (δKO) mice compared to wild type (δWT) mice. δKO tumors are smaller and showed reduced proliferation. DNA sequencing indicated that all δWT tumors had activating mutations in KRAS, whereas only 69% of δKO tumors did, suggesting that PKCδ acts as a tumor promoter downstream of oncogenic KRAS, while acting as a tumor suppressor in other oncogenic contexts. Similar results were obtained in a panel of NSCLC cell lines with oncogenic KRAS, but which differ in their dependence on KRAS for survival. RNAi-mediated attenuation of PKCδ inhibited anchorage-independent growth, invasion, migration and tumorigenesis in KRAS-dependent cells. These effects were associated with suppression of MAPK pathway activation. In contrast, PKCδ attenuation enhanced anchorage-independent growth, invasion and migration in NSCLC cells that were either KRAS-independent or that had wild-type KRAS. Unexpectedly, our studies indicate that the function of PKCδ in tumor cells depends on a specific oncogenic context, as loss of PKCδ in NSCLC cells suppressed transformed growth only in cells dependent upon oncogenic KRAS for proliferation and survival.

PKCd is essential for apoptosis, but regulation of the proapoptotic function of this ubiquitous kinase is not well understood. Nuclear translocation of PKCd is necessary and sufficient to induce apoptosis and is mediated via a Cterminal bipartite nuclear localization sequence. However, PKCd is found predominantly in the cytoplasm of nonapoptotic cells, and the apoptotic signal that activates its nuclear translocation is not known. We show that in salivary epithelial cells, phosphorylation at specific tyrosine residues in the N-terminal regulatory domain directs PKCd to the nucleus where it induces apoptosis. Analysis of each tyrosine residue in PKCd by site-directed mutagenesis identified two residues, Y64 and Y155, as essential for nuclear translocation. Suppression of apoptosis correlated with suppressed nuclear localization of the Y-F mutant proteins. Moreover, a phosphomimetic PKCd Y64D/Y155D mutant accumulated in the nucleus in the absence of an apoptotic signal. Forced nuclear accumulation of PKCd-Y64F and Y155F mutant proteins, by attachment of an SV40 nuclear localization sequence, fully reconstituted their ability to induce apoptosis, indicating that tyrosine phosphorylation per se is not required for apoptosis, but for targeting PKCd to the nucleus. We propose that phosphorylation/dephosphorylation of PKCd in the regulatory domain functions as a switch to promote cell survival or cell death.

Protein Kinase C delta (PKCδ) regulates apoptosis in the mammary gland, however the functional contribution of PKCδ to the development or progression of breast cancer has yet to be determined. Meta-analysis of ErbB2-positive breast cancers shows increased PKCδ expression, and a negative correlation between PKCδ expression and prognosis. Here we present in vivo evidence that PKCδ is essential for the development of mammary gland tumors in a ErbB2-overexpression transgenic mouse model, and in vitro evidence that PKCδ is required for proliferative signaling downstream of the ErbB2 receptor. MMTV-ErbB2 mice lacking PKCδ (δKO) have increased tumor latency compared to MMTV-ErbB2 wild type (δWT) mice, and tumors show a dramatic decrease in Ki-67 staining. To explore the relationship between PKCδ and ErbB2-driven proliferation more directly, we used MCF-10A cells engineered to express a synthetic ligand-inducible form of the ErbB2 receptor. Depletion of PKCδ with shRNA inhibited ligand-induced growth in both 2D (plastic) and 3D (Matrigel) culture, and correlated with decreased phosphorylation of the ErbB2 receptor, reduced activation of Src, and reduced activation of the MAPK/ERK pathway. Similarly, in human breast cancer cell lines in which ErbB2 is overexpressed, depletion of PKCδ suppresses proliferation, Src, and ERK activation. PKCδ appears to drive proliferation through formation of an active ErbB2/PKCδ/Src signaling complex, as depletion of PKCδ disrupts association of Src with the ErbB2 receptor. Taken together, our studies present the first evidence that PKCδ is a critical regulator of ErbB2-mediated tumorigenesis, and suggest further investigation of PKCδ as a target in ErbB2-positive breast cancer.

Background: Tyrosine phosphorylation regulates nuclear translocation of proapoptotic protein kinase C delta (PKC␦). Results: Tyrosine phosphorylation causes a conformational change that exposes the nuclear localization sequence, allowing binding of importin-␣. Conclusion: Nuclear localization of PKC␦ is regulated by access of importin-␣ to the nuclear localization sequence. Significance: Nuclear import of PKC␦, which induces apoptosis, is tightly regulated so as to prevent inappropriate cell death.

Edited by Alex TokerDNA damage-mediated activation of extracellular signalregulated kinase (ERK) can regulate both cell survival and cell death. We show here that ERK activation in this context is biphasic and that early and late activation events are mediated by distinct upstream signals that drive cell survival and apoptosis, respectively. We identified the nuclear kinase mitogensensitive kinase 1 (MSK1) as a downstream target of both early and late ERK activation. We also observed that activation of ERK3 MSK1 up to 4 h after DNA damage depends on epidermal growth factor receptor (EGFR), as EGFR or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/ ERK inhibitors or short hairpin RNA-mediated MSK1 depletion enhanced cell death. This prosurvival response was partially mediated through enhanced DNA repair, as EGFR or MEK/ERK inhibitors delayed DNA damage resolution. In contrast, the second phase of ERK3 MSK1 activation drove apoptosis and required protein kinase C␦ (PKC␦) but not EGFR. Genetic disruption of PKC␦ reduced ERK activation in an in vivo irradiation model, as did short hairpin RNA-mediated depletion of PKC␦ in vitro. In both models, PKC␦ inhibition preferentially suppressed late activation of ERK. We have shown previously that nuclear localization of PKC␦ is necessary and sufficient for apoptosis. Here we identified a nuclear PKC␦3 ERK3 MSK1 signaling module that regulates apoptosis. We also show that expression of nuclear PKC␦ activates ERK and MSK1, that ERK activation is required for MSK1 activation, and that both ERK and MSK1 activation are required for apoptosis. Our findings suggest that location-specific activation by distinct upstream regulators may enable distinct functional outputs from common signaling pathways. . 3 The abbreviations used are: IR, irradiation; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; shRNA, short hairpin RNA; SCR, scrambled; EGFR, epidermal growth factor receptor; NLS, nuclear localization sequence; DUSP, dual specificity phosphatase; Gy, gray; m.o.i., multiplicity of infection. cro ARTICLE 4488

Recent studies suggest that the presence of a KRAS mutation may be insufficient for defining a clinically homogenous molecular group, as many KRAS mutant tumors lose reliance on K-Ras for survival. Identifying pathways that support K-Ras dependency may define clinically relevant KRAS sub-groups and lead to the identification of new drug targets. We have analyzed a panel of 17 KRAS mutant lung cancer cell lines classified as K-Ras dependent or independent, for co-dependency on PKCδ. We show that functional dependency on K-Ras and PKCδ co-segregate, and that dependency correlates with a more epithelial-like phenotype. Furthermore, we show that the pro-apoptotic and pro-tumorigenic functions of PKCδ also segregate based on K-Ras dependency, as K-Ras independent cells are more sensitive to topoisomerase inhibitors, and depletion of PKCδ in this sub-group suppresses apoptosis through increased activation of ERK. In contrast, K-Ras dependent lung cancer cells are largely insensitive to topoisomerase inhibitors, and depletion of PKCδ can increase apoptosis and decrease activation of ERK in this sub-group. We have previously shown that nuclear translocation of PKCδ is necessary and sufficient for pro-apoptotic signaling. Our current studies show that K-Ras dependent cells are refractive to PKCδ driven apoptosis. Analysis of this sub-group showed increased PKCδ expression and an increase in the nuclear:cytoplasmic ratio of PKCδ. In addition, targeting PKCδ to the nucleus induces apoptosis in K-Ras independent, but not K-Ras dependent NSCLC cells. Our studies provide tools for identification of the subset of patients with KRAS mutant tumors most amenable to targeting of the K-Ras pathway, and identify PKCδ as a potential target in this tumor population. These sub-groups are likely to be of clinical relevance, as high PKCδ expression correlates with increased overall survival and a more epithelial tumor phenotype in patients with KRAS mutant lung adenocarcinomas.

We have previously shown that Protein Kinase C delta (PKCδ) functions as a tumor promoter in non-small cell lung cancer (NSCLC), specifically in the context of K-ras addiction. Here we define a novel PKCδ -> integrin αVβ3->Extracellular signal-Regulated Kinase (ERK) pathway that regulates the transformed growth of K-ras dependent NSCLC cells. To explore how PKCδ regulates tumorigenesis, we performed mRNA expression analysis in four KRAS mutant NSCLC cell lines that stably express scrambled shRNA or a PKCδ targeted shRNA. Analysis of PKCδ-dependent mRNA expression identified 3183 regulated genes, 210 of which were specifically regulated in K-ras dependent cells. Genes that regulate extracellular matrix and focal adhesion pathways were most highly represented in this later group. In particular, expression of the integrin pair, αVβ3, was specifically reduced in K-ras dependent cells with depletion of PKCδ, and correlated with reduced ERK activation and reduced transformed growth as assayed by clonogenic survival. Re-expression of PKCδ restored ITGAV and ITGB3 mRNA expression, ERK activation and transformed growth, and this could be blocked by pretreatment with a αVβ3 function-blocking antibody, demonstrating a requirement for integrin αVβ3 downstream of PKCδ. Similarly, expression of integrin αV restored ERK activation and transformed growth in PKCδ depleted cells, and this could also be inhibited by pretreatment with PD98059. Our studies demonstrate an essential role for αVβ3 and ERK signalingdownstream of PKCδ in regulating the survival of K-ras dependent NSCLC cells, and identify PKCδ as a novel therapeutic target for the subset of NSCLC patients with K-ras dependent tumors.