Novel Functions of the Phospholipase D2-Phox Homology Domain in Protein Kinase Cζ Activation

Abstract

It has been established that protein kinase C (PKC) participates in diverse signaling pathways and cellular functions in a wide variety of cells, exhibiting properties relevant to cellular survival and proliferation. Currently, however, the regulation mechanism of PKC remains elusive. Here, for the first time, we determine that phospholipase D2 (PLD2) enhances PKC activity through direct interaction in a lipase activity-independent manner. This interaction of the PLD2-Phox homology (PX) domain with the PKC-kinase domain also induces the activation loop phosphorylation of PKC and downstream signal stimulation, as measured by p70 S6 kinase phosphorylation. Furthermore, only the PLD2-PX domain directly stimulates PKC activity in vitro, and it is necessary for the formation of the ternary complex with phosphoinositide-dependent kinase 1 and PKC. The mutant that substitutes the triple lysine residues (Lys 101 , Lys 102, and Lys 103) within the PLD2-PX domain with alanine abolishes interaction with the PKC-kinase domain and activation of PKC. Moreover, breast cancer cell viability is significantly affected by PLD2 silencing. Taken together, these results suggest that the PLD2-mediated PKC activation is induced by its PX domain performing both direct activation of PKC and assistance of activation loop phosphorylation. Furthermore, we find it is an important factor in the survival of breast cancer cells.Protein kinase C (PKC) has been implicated in many cellular key functions, such as cell proliferation, survival, and migration (2, 40, 44). The PKC family is subclassified into three groupsclassical, novel, and atypical PKC-according to differences in the lipid activation profile (42). It has been established that the phosphorylation and activation of atypical PKC, especially, is an important factor in the survival of cancer cells (21, 41). The phosphorylation of PKC is one of the main mechanisms for regulating its activity. Recently, it has been reported that moderate activation of PKC is mediated through activation loop phosphorylation by phosphoinositide-dependent kinase 1 (PDK-1), followed by a subsequent autophosphorylation (8,38,59). PKC is also stimulated by the interaction of acidic lipids, including phosphatidic acid (PA) and phosphoinositides. Due to its structural uniqueness, PKC is insensitive to second messengers, such as Ca 2ϩ or diacylglycerol (DAG), known to be potent activators of the other families (46). Therefore, the activation of PKC may be expected to rely on a peculiar mechanism, which is perhaps regulated by many cellular proteins. However, the exact protein-protein interactions intrinsic to the regulation mechanism of PKC remain largely unclear.Phospholipase D (PLD) exists as a membrane-bound protein and is widely distributed in a variety of cells. It hydrolyzes phosphatidylcholine to generate choline and PA as a response to diverse stimuli. In many cancer cells, the abnormal overexpression of PLD is associated with the promotion of mitogenesis, oncogenic transformation, and cell proliferation an...

Phosphatidylinositol (3,4,5)-trisphosphate specifically interacts with the phox homology domain of phospholipase D1 and stimulates its activity

“…Transfection was carried out with LipofectAMINE, according to the manufacturer's instructions. In HEK 293 cells, depletion of endogenous PLD2 was performed using small interference RNA (siRNA) for PLD2, as described previously (Kim et al, 2005). After starving the cells for 24 hours, they were incubated with 10 Ci [ 3 H]myristic acid for 3 hours.…”

Protein Kinase Cζ Abrogates the Proapoptotic Function of Bax through Phosphorylation

“…The mechanisms for activation have been established for sequential phosphorylation, the recruiting of proper localization, and the exchanging of binding proteins (11). Growing evidence indicates that PKC family members play important roles in regulating cell survival and apoptosis (12)(13)(14)(15).…”

“…PKC is insensitive to second messengers such as Ca 2ϩ and diacylglycerol. Thus, its activity is primarily regulated by protein-protein interaction and phosphorylation (11). The signal mechanisms for PKC activation include the PDK1-dependent (i.e.…”

Phospholipase D2 mediates signaling by ATPase class I type 8B membrane 1

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