
Various extracellular information appears to be passed into the cell by various mechanisms of transmembrane signaling (1) . As the inner surface of the plasma membrane is always in contact with cytoplasmic proteins, it has been expected that there must be some sort of direct interaction between them. Recently we have reported that several cytoplasmic proteins associate with phospholipid vesicles (liposomes) (23) and that some cytoplasmic proteins, such as the 33 kDa protein, from PMNs associated with PS-containing liposomal membrane in a calcium-dependent manner (20, 23, 24) . This membrane-protein interaction is quite similar to that between Ca2+-and phospholipid-dependent protein kinase (protein kinase C) and the plasma membrane (17, 18). Furthermore, it is reported that the association of protein kinase C with the plasma membrane results in activation of the enzyme (6, 16, 25), and the characteristic structure of vesicles formed from various phospholipids affects protein kinase C activity (2) . It was also found by an immunocytochemical method (20) that tumor-promoting 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused a rapid translocation of protein kinase C from the cytoplasm to plasma membrane. Similarly, translocation of the 33 kDa protein from the cytoplasm to plasma membrane was caused by formylmethionyl-leucyl-phenylalanine (FMLP) (20). These results suggest that the interaction between cytoplasmic proteins and the inner surface of the plasma membrane may participate in regulating cellular signal transduction. Moreover, Ca2+-dependent association of these cytoplasmic proteins similar to that with PScontaining liposomes was observed with CL-containing liposomes (25) . Therefore, it is proposed that the activation of protein kinase C may be caused by the association of this enzyme with CL-containing phospholipid liposomes. Similar phospholipid dependencies of TPA binding and of the activation of the enzyme have been observed with various anionic phospholipids (PS, CL, phosphatidic acid) (7, 8). However, very little has been known about the detailed biochemical mechanism of CL-induced enzyme activation. In this paper we described the interaction of CL-activated protein kinase C with the membrane and report that the membrane-associated cytoplasmic proteins were phosphorylated preferentially by the membrane-bound enzyme. 
RESULTS

AND DISCUSSION
Association of C-kinase with CL-containing liposomes. It was recently demonstrated that multifunctional Ca2+-binding proteins undergo a substantial conformational change upon binding to Ca2+, exposing hydrophobic sites (4, 5, 25). The binding of protein kinase C to a specific site in biological membrane, such as PS, was induced by micromolar concentrations of Ca2+ and resulted in activation of the enzyme (6, 25). As reported in previous papers (20, 23), when the PMN supernatant was incubated with the phospholipid liposomes which contain CL, several proteins, including the 33 kDa protein, associated with the liposomes in a calcium-dependent manner, and then were separated from non-associated proteins by using the flotation method ( Fig. 1) (22) . In this case, only the liposomal fraction (Fig. 2a, L) showed the activity of protein kinase C, and no enzyme activity was observed in the non-associated fraction in the absence of exogenous substrate (H1 histone) ( Fig. 2A, R) . Furthermore, the phosphorylation of the liposome-associated proteins was inhibited by mixing with the non-associated fraction ( Fig. 2A, L+R) C-kinase activity was detected in either rat liver mitochondria or E. coli. At present, there is no evidence of a role of CL in mitochondria and E. coli in relation to the enzyme activity in vivo (data not shown). Stimulation of neutrophils by external agents elicits a variety of responses. It has been proposed that many of these responses are mediated by a series of events involving the tight binding of protein kinase C to the plasma membrane, resulting in its activation and phosphorylation of membrane proteins (17).
Together with previous results (19, 23), the data obtained in these experiments support the idea that the signal, which translocates protein kinase C from cytoplasm to plasma membrane by exposing its hydrophobic domain by PMA or mobilized intracellular calcium ions, would lead to phosphorylation of not only the membrane protein but also the transiently-associated cytoplasmic proteins which regulate the state of membrane lipids or the intracellular calcium ion concentration. Therefore, different cytoplasmic proteins may be phosphorylated by different stimuli (such as FMLP and PMA). The experimental conditions were the same as described in Fig. 2 . The 32P-labeled proteins were observed in SDS-PAGE (10% polyacrylamide gel) by autoradiography.
A) Association with DPPC/Chol/CL liposomes; B) denatured protein after association with DPPC/Chol/CL liposomes; C) Rat brain protein kinase C was added to B; PKC, protein kinase C; L, liposome-associated fraction; R, non-liposome associated fraction.

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