Understanding the intracellular mechanisms by which binding of ligands, such as hormones and growth factors, to their specific receptors elicits the appropriate cellular response has long been a topic of great interest. Considerable excitement was generated when it was recognised that several receptor-ligand interactions operate via the hydrolysis of inositol phospholipids. This yields, at least, two 'second messengers', namely, inositol 1,4,5-trisphosphate [Ins(l ,4,5)P3], which causes the release of Ca2 The pertinent question that follows is, how do PKC activation and elevation of the intracellular Ca2 ' concentration evoke cell responses? In this review, attention has been focussed on PKC, and the consequences of its activation in resting human T cells. Evidence that PKC activity is, at least partially, responsible for activation of resting human T cells will be examined, and some of the more recent research investigating how PKC activation elicits this cell response will be described. ACTIVATION OF RESTING HUMAN T CELLS T cell activationBefore beginning to discuss the signal transduction mechanisms involved, the phenomenon which, in this review, constitutes 'T cell activation' should be described. Appropriate stimulation of T cells results in the transcription of over fifty genes, leading to the expression of a variety of molecules (reviewed in [3]). These include interleukin-2 (IL-2) and a 55-kDa polypeptide which combines with a constitutively expressed 70 -75-kDa polypeptide [4, 51, to form the highaffinity interleukin receptor reviewed in [6]). Expression of the 55-kDa polypeptide (IL-2Ra) and secretion of IL-2 is an identifiable activation state in which T cells are irreversibly committed to proliferation, for autocrine binding of IL-2 to the high-affinity 1L-2R triggers DNA synthesis and completion of the mitotic cycle [7, 81. Activation can be determined, therefore, by IL-2Rct expression and secretion of IL-2, or, since these molecules are controlled at the level of transcription [9, lo], by appearance of the mRNA for IL-2Ra or IL-2. Proliferation can be used as an indirect measurement of expression of the high-affinity IL-2R and secretion of IL-L .

Purified resting human T cells can be induced to express the a subunit of the interleukin 2 receptor and to proliferate by treatment with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-0-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the a subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.In several cell types, activation of cell responses results from the receptor-mediated hydrolysis of inositol phospholipids, generating two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, which activate protein kinase C (PKC) and elevate the intracellular Ca2+ concentration, respectively (reviewed in refs. 1 and 2). Antigenic activation of resting human T cells can be mimicked by the synergistic action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates PKC, and the calcium ionophore, ionomycin, which raises the intracellular Ca2+ concentration (3). This treatment results in the secretion of interleukin 2 (IL-2) and expression of a 55-kDa a subunit, which combines with a constitutively expressed 70-kDa ,B subunit (4, 5) to form the high-affinity IL-2 receptor (IL-2R; reviewed in ref. 6). Both expression of the a subunit of IL-2R (IL-2Ra) and secretion of IL-2 are controlled at the level of gene transcription (7) and represent a state of irreversible activation ofT cells, because autocrine binding of IL-2 to its cell surface receptor triggers proliferation (8).It was recently demonstrated that, in a >991% pure population of resting human T cells, replacing TPA with membrane-permeable DAGs, which activate PKC in a more physiological manner than TPA, does not induce expression ofthe IL-2Ra or cell proliferation (9). One explanation for the difference in cell response to TPA or DAGs is that TPA stimulates prolonged (longer than 1 hr) activation of PKC (10), whereas DAGs are rapidly converted to the corresponding phosphatidic acid by DAG kinase (11). In the present study, the duration of PKC activation by TPA or by the DAG 1,2-dioctanoylglycerol (DiC8) Chemical (Osaka), trypsin treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone was purchased from Worthington, and soybean trypsin inhibitor was from Sigma. MBP4_14 (GlnLys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu), synthesized in this laboratory, is a PKC-specific substrate (A.K. and Y.N., unpublished observations).Preparation of Pure T Lymphocytes. A population of resting T cells was purified from human peripheral ...

The down-regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre-B, pre-T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly /III) and type III (a), upon hydroxyapatite column chromatography.Biochemical and immunocytochemical analysis revealed that, when these cells were treated with l2-O-tetradecanoylphorbol I3-acetate (TPA), the time course of down-regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.

Using a preparation of purified human T lymphocytes, we were able to resolve a partially purified protein kinase C (PKC) enzyme fraction into two distinct subspecies, of approximately equal activity. Biochemical and immunocytochemical analysis revealed that these fractions closely resembled the type II(B) and type III(a) PKC subspecies previously identified and characterised from brain tissue. These results provide valuable information for further studies on the role of individual PKC subspecies in T lymphocyte proliferation.

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