Phosphorylation sites of protein kinase C δ in H ₂ O ₂ -treated cells and its activation by tyrosine kinase in vitro

Abstract

Protein kinase C ␦ (PKC ␦) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H2O2, the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC ␦ in the H2O2-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC ␦ in response to H2O2. P rotein kinase C (PKC) comprises a family of more than ten serine͞threonine protein kinases that are involved in a variety of signal transduction pathways (1). Each isoform has the regulatory and catalytic domains in the amino-and carboxylterminal halves, respectively. The isoforms are divided into three groups, cPKC, nPKC, and aPKC, because of the structural differences in their regulatory domains. The cPKC and nPKC isoforms are activated by diacylglycerol produced from receptormediated hydrolysis of inositol phospholipids and are the prime targets of tumor-promoting phorbol esters that bind to the cysteine-rich sequence, named the C1 region, in the regulatory domain. In general, a number of protein kinases are known to be controlled by phosphorylation (2), and the PKC family members have three phosphorylation motif sites mostly conserved among the family (3). One is a threonine residue in the activation loop of the catalytic domain, and the others are serine and threonine residues located in the carboxyl-terminal end region, named the turn and hydrophobic motifs, respectively.The PKC isoforms are further phosphorylated on tyrosine upon stimulation of the cells (4), and the role of tyrosine phosphorylation has been investigated for PKC ␦, a member of the nPKC group (5). That is, PKC ␦ is phosphorylated on tyrosine in v-ras-transformed keratinocytes (6) and in various cells stimulated with phorbol ester, growth factors, and hormones (7-14). However, controversial results are reported on the functional consequence of the tyrosine phosphorylation reaction induced by these membrane-coupled signaling processes. In keratinocytes, tyrosine phosphorylation reduces its catalytic activity (6, 12), whereas in other cases the modification reaction enhances the enzymatic activity (4, 7, 8) or even alters...

Rottlerin protected dopaminergic cell line from cytotoxicity of 6-hydroxydopamine by inhibiting PKCδ phosphorylation

“…However, controversy still exists concerning the most important mode of PKC activation [3,4,16,18,19,[28][29][30] . Recent research has suggested that PKCδ phosphorylation at the site of Tyr311 could regulate the proteolytic activation and proapoptotic function of PKCδ in dopaminergic neuronal cells [28] .…”

“…However, some researchers have suggested that phosphorylation at the site of Thr505 in PKCδ might just lead to the allosteric effect, which is one of the prerequisites for PKCδ activation [19] , and the proteolysis of PKCδ by caspase-3 represents the terminal pattern of PKCδ activation. The accumulation of PKCδ catalytic fragment is an important event in cell apoptosis [18] , particularly in H 2 O 2 -or MPP + -induced cytotoxic responses [29,30] . Our studies demonstrate that 6-OHDA could induce PKCδ phosphorylation at the site of Thr505 in PC12 cells (Fig.…”

Specific protein kinase C isoforms as transducers and modulators of insulin signaling

“…Tyrosine phosphorylation of PKCδ has been reported for responses of salivary gland cells in response to carbachol [44,45], COS-7 cells in response to H 2 O 2 [46,47], and, in addition, skeletal muscle cells in response to insulin [12]. Mouse, rat and human PKCδ contain 19, 21 and 20 tyrosine residues respectively.…”

“…In current studies, we (unpublished) have found that PKCε is also tyrosine phosphorylated and activated in response to insulin. Src tyrosine kinase has been shown to be involved in insulin and other growth factor signaling in several cell types, including skeletal muscle [52][53][54][55][56][57][58][59][60][42][43][44][45][46][47][48][49]. Moreover, in the case of PKCs α and δ, insulin-induced tyrosine phosphorylation is apparently mediated by the Src family of tyrosine kinases, if not Src tyrosine kinase itself ( [31] and unpublished).…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…It has been reported that Src-dependent phosphorylation of tyrosine residues on PKCs catalytic domain can augment its activity. Although in vitro studies have shown that multiple PKCs can be activated in this manner [87], the only member of the PKC family that has been shown to be tyrosine phosphorylated in vivo in VSM cells is PKCδ [31]. PKCδ has multiple tyrosine residues on its regulatory and catalytic domain and in vivo studies have reported that phosphorylation of the tyrosine residues occur exclusively in the regulatory domain [89].…”