The pleckstrin homology domain of protein kinase D interacts preferentially with the eta isoform of protein kinase C

The results presented here demonstrate that protein kinase D (PKD) and PKCeta transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCeta. The presence of PKCeta in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCeta was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCepsilon, and did not bind PKCzeta. When PKCeta was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCeta immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCeta. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCeta. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs epsilon and eta, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.     

Protein kinase C phosphorylates protein kinase D activation loop Ser744 and Ser748 and releases autoinhibition by the pleckstrin homology domain

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser(744) and Ser(748) located in the catalytic domain activation loop, but whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is unclear. Here, we explore the mechanism whereby PKC activates PKD in response to cellular stimuli. We first assessed in vitro PKC-PKD transphosphorylation and PKD activation. A PKD738-753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their known substrate specificities. A similar peptide with glutamic acid replacing Ser(748) was preferentially phosphorylated by PKCepsilon, suggesting that PKD containing phosphate at Ser(748) is rapidly targeted by this isoform at Ser(744). When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser(748). In contrast, addition of purified PKCepsilon to the incubation mixture induced rapid Ser(744) and Ser(748) phosphorylation, concomitant with persistent 2-3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domain-mediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-deltaPH was not abrogated by mutation of Ser(744) and Ser(748) to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKCepsilon-PKD phosphorylation cascade and provide additional insight into the activation mechanism.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation

Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin α(IIb)β(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.     

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.     

Novel functions of the phospholipase D2-Phox homology domain in protein kinase Czeta activation

It has been established that protein kinase Czeta (PKCzeta) participates in diverse signaling pathways and cellular functions in a wide variety of cells, exhibiting properties relevant to cellular survival and proliferation. Currently, however, the regulation mechanism of PKCzeta remains elusive. Here, for the first time, we determine that phospholipase D2 (PLD2) enhances PKCzeta activity through direct interaction in a lipase activity-independent manner. This interaction of the PLD2-Phox homology (PX) domain with the PKCzeta-kinase domain also induces the activation loop phosphorylation of PKCzeta and downstream signal stimulation, as measured by p70 S6 kinase phosphorylation. Furthermore, only the PLD2-PX domain directly stimulates PKCzeta activity in vitro, and it is necessary for the formation of the ternary complex with phosphoinositide-dependent kinase 1 and PKCzeta. The mutant that substitutes the triple lysine residues (Lys101, Lys102, and Lys103) within the PLD2-PX domain with alanine abolishes interaction with the PKCzeta-kinase domain and activation of PKCzeta. Moreover, breast cancer cell viability is significantly affected by PLD2 silencing. Taken together, these results suggest that the PLD2-mediated PKCzeta activation is induced by its PX domain performing both direct activation of PKCzeta and assistance of activation loop phosphorylation. Furthermore, we find it is an important factor in the survival of breast cancer cells.     

PH结构域研究进展

ＰＨ结构域是一种新发现的约由１００￣１２０个氨基酸残基组成的功能性结构域,广泛分布于单细胞生物和无脊椎、脊椎动物及人的细胞骨架蛋白和信号分子等１００多种蛋白中,能与脂类、Ｇ蛋白的βγ亚单位、ＰＫＣ等配体相结合,推测其对蛋白质具有细胞和亚细胞水平的膜定位作用,并参与调节宿主蛋白 活性,从而介导信号转导过程中的蛋白南－脂类、蛋白质－蛋白质之间的相互作用。     

A novel Arabidopsis thaliana dynamin-like protein containing the pleckstrin homology domain

A full-length cDNA encoding a novel type of plant dynamin-like protein, ADL3, was isolated from Arabidopsis thaliana. ADL3 is a high molecular weight GTPase whose GTP-binding domain shows a low homology to those of other plant dynamin-like proteins. ADL3 contains the pleckstrin homology domain as is in mammalian dynamins, although other plant dynamin-like proteins reported lack this domain. The ADL3 gene was expressed weakly in various tissues, except for siliques with high level expression, which is distinct from the case for other plant dynamin-like protein genes. Taken together, it is predicted that the mode of activation of ADL3 is different from those of other plant homologues.     

The pleckstrin homology domain: An intriguing multifunctional protein module

Pleckstrin homology (PH) domains are a family of compact protein modules defined by sequences of roughly 100 amino acids. These domains are common in vertebrate, Drosophila, C. elegans and yeast proteins, suggesting an early origin and fundamental importance to eukaryotic biology. Many enzymes which have important regulatory functions contain PH domains, and mutant forms of several such proteins are implicated in oncogenesis and developmental disorders. Numerous recent studies show that PH domains bind various proteins and inositolphosphates. Here I discuss PH domains in detail and conclude that they form a versatile family of membrane binding and protein localization modules.     

Tyrosine phosphorylation regulates the proteolytic activation of protein kinase Cdelta in dopaminergic neuronal cells

Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-delta (PKCdelta) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCdelta can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H(2)O(2)(0-300 microm) induced a dose-dependent increase in cytotoxicity, caspase-3 activation and PKCdelta cleavage. H(2)O(2)-induced proteolytic activation of PKC was delta mediated by the activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 microm) and the p60(Src) tyrosine-specific kinase inhibitor (TSKI; 5 microm) dramatically inhibited H(2)O(2) and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCdelta cleavage, kinase activation, and apoptotic cell death. H(2)O(2) treatment also increased phosphorylation of PKCdelta at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCdelta(Y311F) mutant protein exhibited resistance to H(2)O(2)-induced PKCdelta cleavage, caspase activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCdelta can regulate the proteolytic activation and proapoptotic function of the kinase in dopaminergic neuronal cells.     

High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate

The products of PI 3-kinase activation, PtdIns(3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2, trigger physiological processes, by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized PtdIns(3,4,5)P3/PtdIns(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent pro-tein kinase-1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P3/PtdIns(3,4)P2 binding PH domain. Here, we describe the high-resolution crystal structure of the isolated PH domain of PKB(alpha) in complex with the head group of PtdIns(3,4,5)P3. The head group has a significantly different orientation and location compared to other Ins(1,3,4,5)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2.     

Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain

The N-terminus of ceramide kinase (CERK) is thought to be myristoylated and to contain a pleckstrin homology (PH) domain. We found that deletion of this region (DeltaPH-CERK) ablates activity. This is not due to prevention of N-terminal myristoylation since a G2A CERK mutant, which cannot be myristoylated, was active. CERK was able to bind liposomes, as well as the isolated unmyristoylated PH domain; DeltaPH-CERK was not. Upon analysis of EGFP-tagged proteins, CERK was found associated with the Golgi complex. Osmotic cell swelling induced translocation of CERK to the plasma membrane and formation of large vesicles displaying bound CERK. None of these features occurred with DeltaPH-CERK, which remained disseminated throughout the cytoplasm. These findings show that the PH domain of CERK is essential for localization, translocation, and activity of this lipid kinase.     

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.     

A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.     

Ceramide disables 3-phosphoinositide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by a PKCzeta-dependent mechanism

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKCzeta, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCzeta interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKCzeta, leading to phosphorylation of the PKB-PH domain on Thr(34). This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP(3) even in the presence of a ceramide-activated PKCzeta and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCzeta and a kinase-dead PKCzeta both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKCzeta-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.     

Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.

In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.     

Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis

The GTPase dynamin plays an essential role in clathrin-mediated endocytosis [1] [2] [3]. Substantial evidence suggests that dynamin oligomerisation around the necks of endocytosing vesicles and subsequent dynamin-catalysed GTP hydrolysis is responsible for membrane fission [4] [5]. The pleckstrin homology (PH) domain of dynamin has previously been shown to interact with phosphoinositides, but it has not been determined whether this interaction is essential for dynamin's function in endocytosis [6] [7] [8] [9]. In this study, we address the in vivo function of the PH domain of dynamin by assaying the effects of deletions and point mutations in this region on transferrin uptake in COS-7 fibroblasts. Overexpression of a dynamin construct lacking its entire PH domain potently blocked transferrin uptake, as did overexpression of a dynamin construct containing a mutation in the first variable loop of the PH domain. Structural modelling of this latter mutant suggested that the lysine residue at position 535 (Lys535) may be critical in the coordination of phosphoinositides, and indeed, the purified mutant no longer interacted with lipid nanotubes. Interestingly, the inhibitory phenotype of cells expressing this dynamin mutant was partially relieved by a second mutation in the carboxy-terminal proline-rich domain (PRD), one that prevents dynamin from binding to the Src homology 3 (SH3) domain of amphiphysin. These data demonstrate that dynamin's interaction with phosphoinositides through its PH domain is essential for endocytosis. These findings also support our hypothesis that PRD-SH3 domain interactions are important in the recruitment of dynamin to sites of endocytosis.