Novel Roles of Specific Isoforms of Protein Kinase C in Activation of the c-fos Serum Response Element

Protein kinase C (PKC) is a family of phospholipid-dependent serine/threonine kinases which can be activated upon external stimulation of cells by various ligands including growth factors, hormones, and neurotransmitters (7, 51). Specific isoforms of PKC can be activated by calcium, various phospholipids, diacylglycerol (DAG) generated from phospholipase C (PLC) or PLD, and fatty acids generated from PLA2, depending on the PKC isoforms (7, 17, 32, 36). Molecular cloning has identified 11 distinct isoforms of PKC in mammalian cells. Based on their structure, these isoforms are divided into three groups: (i) classical PKCs (α, βI, βII, γ), which can be activated by DAG or calcium; (ii) novel PKCs (δ, ɛ, η, θ, μ), which can be activated by DAG but not by calcium; and (iii) atypical PKCs (ζ, ι), which are not responsive to either DAG or calcium. Each of these isoforms contain an amino-terminal regulatory domain and a carboxy-terminal catalytic kinase domain. A number of studies have shown that the activation of cellular PKC by the potent phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-ester (TPA) induces the expression of several immediate response genes including c-fos. The specific signaling pathways involved in this process are not known with certainty. In addition, since most cell types contain multiple isoforms of PKC and since there are no isoform-specific inhibitors of PKC, it is not certain which isoforms of PKC mediate these responses. With respect to the above issues, the induced expression of the immediate-early response gene c-fos is of particular interest since this induction is usually rapid, within 30 min, and transient in various cell types following exposure to TPA, various growth factors, neurotrophins, or neurotransmitters. Furthermore, the signal transduction pathways leading to activation of the c-fos promoter have been studied in great detail and have served as an excellent model for studying the biochemical mechanisms by which extracellular signals generated at the plasma membrane activate gene transcription (30, 31) (also see Fig. ​Fig.6).6). The serum response element (SRE) in the c-fos promoter is necessary and sufficient for rapid induction of the c-fos gene by serum, growth factors and TPA (29, 61). Two transcription factors, serum response factor (SRF) and ternary complex factor (TCF), bind to the SRE and mediate transcriptional activation. SRF is a ubiquitously expressed transcription factor that binds as a dimer to the CArG box of the c-fos SRE. It is a 67-kDa protein with a central core that contains the DNA binding and dimerization domains. SRF also has a C-terminal transcriptional activation domain and an N-terminal domain that can be phosphorylated by casein kinase II (CKII) and Ca2+/calmodulin-dependent kinase (CaMK) (40, 41, 47). SRF forms a ternary complex with TCF on the SRE. In this ternary complex, TCF interacts with both the dimerization domain of SRF and a purine-rich sequence (CAGGAT) at the 5′ end of the SRE. TCF interacts with the c-fos SRE only if the SRE is already occupied by SRF. TCF is encoded by a family of ets-related genes which includes the genes encoding Elk-1, SRF accessory protein 1 (SAP-1), and SAP-2. The TCFs have three conserved regions, referred to as the A, B, and C boxes. The A box is the N-terminal ets-related DNA binding domain. The B box is the SRF binding domain. The A and B boxes are necessary and sufficient for ternary-complex formation with SRF on the SRE. The C box is the C-terminal transcriptional activation domain and contains a cluster of serine residues. Phosphorylation of TCF causes increased DNA binding, ternary-complex formation, and transcriptional activation (43, 60). TCF is phosphorylated by at least three major mitogen-activated protein (MAP) kinases, including ERK1/2, JNK, and p38. Serum, growth factors, and TPA induce the phosphorylation of Elk-1/SAP-1 through the Raf-MEK-ERK pathway (19, 26), whereas interleukin-1, tumor necrosis factor alpha, osmotic stress, H2O2, UV radiation, or anisomycin induce phosphorylation of TCF through the MEKK-SEK1-JNK (12, 20, 59, 66) or TAK1-MKK3-p38 pathways (27, 55, 67, 70). Mutants with mutations in the SRE that cannot bind TCF are not responsive to these MAP kinase pathways but remain responsive to serum induction through a TCF-independent pathway that requires SRF (28). In the absence of TCF, SRF can also mediate transcriptional activation by the serum mitogen lysophosphatidic acid (LPA) and also by intracellular activation of heterotrimeric G proteins by aluminum fluoride ion (AlF4−) (23, 50, 54). Functional rhoA is required for serum-, LPA-, and AlF4−-induced transcriptional activation of SRE by SRF, and two other small GTPase rho family members, rac1 and cdc42Hs, also potentiate SRF activity (24, 50). FIG. 6 Proposed signal transduction pathways involved in PKC-mediated SRE activation. Various external stimuli can lead to activation of the three indicated MAP kinase (MAPK) pathways (p38, ERK, and JNK) and also the rhoA pathway. These, in turn, lead to activation ... The precise roles of individual isoforms of PKC in the above-described signaling pathways that lead to activation of the SRE have not been elucidated. This was the goal of the present study. Our strategy was to introduce an SRE-luciferase reporter into NIH 3T3 mouse fibroblasts and to coexpress wild-type or various mutant forms of specific isoforms of PKC and/or dominant negative forms of components of these pathways. We present evidence that in this cell system PKC-α and PKC-ɛ are the major isoforms of PKC that play a role in activation of the SRE and that these PKC isoforms activate not only TCF through both the ERK and JNK pathways but also SRF through the rhoA pathway.