Expression and Biochemical Characterization of Human Protein Kinase C‐θ

Abstract

In this study, the recently identified human protein kinase C-6 (PKC-8) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC-O identified an 80-kDa protein in all human T-cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U-937 cells, but not in a B-lymphoma line (Raji) or in several melanoma, carcinoma, schwanoma or astrocytoma lines, confirming, at the protein level, its predominant expression in hematopoietic cell lines, in particular T cells. Immunoreactive PKC-8 was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, however, caused rapid translocation to the membrane. In order to compare the properties of PKC-O with a representative member of the Ca2+-dependent PKC enzymes, full-length cDNAs encoding PKC-8 or PKC-a were transiently expressed in COS-1 cells, and recombinant enzymes were partially purified via a six-histidine peptide tag. The catalytic activity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differences were found with respect to activation requirements and substrate preferences between PKC-8 and PKC-a. Both enzymes were stimulated by phospholipid and phorbol ester, and were active towards a PKC-derived substrate peptide corresponding to the pseudosubstrate site of PKC. In contrast to PKC-a, however, full activation of PKC-8 did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelinbasic-protein-(MBP)-derived peptide, which was readily phosphorylated by PKC-a, was a poor substrate for PKC-8. Similar to PKC-a, transient PKC-8 overexpression in murine EL4 thymoma cells caused an approximately 2.5-fold increase in the phorbo1-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-8 suggest that it may play a specialized role in T-cell signaling pathways.Molecular cloning and biochemical studies identified protein kinase C (PKC) enzymes as members of a distinct family of serinehhreonine protein kinases that constitutes, at present, ten mammalian members, i.e. a, pI, P I , y, 6, E , [, q, O and z/A. These phospholipid-dependent, diacylglycerolactivated enzymes are thought to play key roles in the regulation of many cellular functions, including proliferation and differentiation (reviewed in [l]). PKC enzymes can be grouped into three subfamilies based on their domain structure. The first subfamily includes the Ca2+-dependent a, p and 'J isoforms [2-61. These enzymes contain, in addition to the catalytic C3 domain that is shared by all members of the family, a phospholipid-binding and diacylglycerob'phorbolester-binding C1 domain and a Ca"-binding C2 domain.PKC isoforms 6, E , q [7-111 and O [12-141 contain the C1

Protein Kinase C-θ Phosphorylation of Moesin in the Actin-binding Sequence

“…Our data demonstrating a minimal contribution of DAG to the PS activation of PKC-phosphorylation of moesin is consistent with the findings of Baier et al (29), who showed a similar minimal effect of phorbol 12-myristate 13-acetate to the PS activation of PKC-. A comparable pattern has been observed with PKC-␦ phosphorylation of the ␥ chain of the IgE receptor, where the rate of threonine phosphorylation was increased 2.5-fold using PI compared with PS, whereas the effect of PG was not tested (53).…”

“…8, maximal kinase activity was obtained using PG and His 6 -moesin, whereas the kinase activity toward MBP was lipidindependent, and histone H1 phosphorylation was minimal. These results are consistent with previous findings of PKCusing an MBP-derived peptide and histone H1 (29).…”

“…Although some isozymes have widespread tissue and cell type distribution (␣, ␦, and ), other isozymes such as PKC-␥, -, and -are more restricted. PKC-is the main novel isozyme expressed in skeletal muscle, testes, platelets, and their megakaryoblastic precursor cells, T-lymphocytes, and neoplastic hemopoietic cells (7,48,49). We have found moesin phosphorylation activity in HL-60 cells, spleen cells, and AML cells, which is consistent with the known distribution of PKC- (29) and in contrast to the tissue distribution of the (50) and the / isoforms, which are not found in spleen (51,52).…”

“…Recombinant His 6 -PKC-Phosphorylates Moesin-To verify that PKC-phosphorylates moesin, we transfected eukaryotic cells with a plasmid bearing PKC-with a His 6 tag at its C terminus (16,29). Jurkat (T-lymphoblastic leukemia) cells were transfected with the His 6 -PKC-plasmid and with the neo vector alone.…”

Altered Composition of the Immunological Synapse in an Anergic, Age-Dependent Memory T Cell Subset

“…Recruitment of PKC-to the membrane is necessary for its activation and involvement in signaling (24). Our group has previously used immunofluorescent methods to show, at the single-cell level, a decline with age in the proportion of CD4 ϩ and CD8 ϩ T cells that mobilize PKC-to the T cell/APC interface in responses triggered by anti-CD3 hybridoma cells (25).…”