Katsuhiko Ase

The common structure and activities of four subspecies of rat brain protein kinase C family

Elucidation of the complete sequences of four cDNA clones (α, βI, βII, and γ) of the rat brain protein kinase C family has revealed their common structure composed of a single polypeptide chain with four constant (C1‐C4) and five variable (V1‐V5) regions. Although these sequences are highly homologous and closely related to one another V3‐, V4‐, and V5‐regions of γ‐subspecies are slightly bigger than the corresponding regions of the other three subspecies. The first constant region, C1, contains a tandem repeat of cysteine‐rich sequence (6, total 12 cysteine residues). The third constant region, C3, has an ATP‐binding sequence which is found in many protein kinases. In adult rat whole brain, the relative activities of α‐, βI, βII, and γ‐subspecies are roughly 16, 8, 55, and 21%, respectively. γ‐Subspecies is expressed after birth apparently only in the central nervous tissue, implying its role in the regulation of specific neuronal functions.

Stereospecificity of diacylglycerol for stimulus-response coupling in platelets☆

In intact platelets, a permeable diacylglycerol having a 1,2-sn- but not 2,3-sn- configuration activated protein kinase C directly. In the presence of Ca2+-ionophore this diacylglycerol caused full activation of platelet release reaction. 1,3-Isomer was inactive. Among these isomers only 1,2-sn-diacylglycerol was converted rapidly to the corresponding phosphatidic acid in both intact and broken cell preparations. Thus, the diacylglycerol which functions in stimulus-response coupling possesses a 1,2-sn-glycerol backbone, and other isomers are not involved in the signal transduction through the protein kinase C pathway.

Isolation of protein kinase C subspecies from a preparation of human T lymphocytes

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(β) and type III(α) 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.

Characterization of protein kinase C from normal and transformed cultured murine fibroblasts

Protein kinase C of normal and ras-transformed NIH 3T3 cells was purified by chromatography on TSK DEAE-5PW, threonine-Sepharose, and TSK phenyl-5PW columns. Comparison of the fibroblast enzyme with several types of rat brain protein kinase C by chromatography on a hydroxyapatite column and by immunoblotting, indicates that both normal and transformed fibroblasts possess only one of the four subspecies of protein kinase C which have been identified in brain tissues. This subspecies presumably has the structure encoded by alpha-sequence or a closely related sequence. No significant difference was seen between those enzymes purified from normal and transformed fibroblasts.

Expression of protein kinase C subspecies in human leukemia- lymphoma cell lines

Expression of protein kinase C (PKC) subspecies was studied in various human leukemia‐lymphoma cell lines. The PKC in most cell lines examined was resolved into two major fractions corresponding to type II (β‐sequence) and type III (α‐sequence) PKC of the rat brain. The amounts of these two subspecies greatly varied among the cell lines. Type I PKC (γ‐sequence) was expressed in none of the cell lines tested, but PKCs with undefined structures were frequently detected. The differential co‐expression of several PKC subspecies is presumably related to the state of cell differentiation.