Investigating the role played by protein-lipid and protein-protein interactions in the membrane association of the p120GAP CaLB domain.

The GTPase activating protein, p120GAP, contains an amino acid sequence motif called the Ca2+-dependent lipid binding domain (CaLB) which mediates a protein-protein interaction between p120GAP and annexin VI and also binds to negatively charged phospholipids. Because membrane association of p120GAP is important for the regulation of p21 Ras activity, we have studied the roles played by Ca2+, phospholipids and annexin VI in the membrane association of p120GAP. Here we demonstrate that a truncated CaLB domain GST fusion protein (GSTGAP618-632), lacking the ability to bind to phospholipids, is able to bind to rat fibroblast membranes in a Ca2+- and concentration-dependent manner. In addition, this fusion protein also binds to annexin VI in an amino acid sequence specific but Ca2+ independent manner. Also, when bound to annexin VI in the presence of Ca2+, this fusion protein has the ability to co-bind to phosphatidylserine vesicles. Thus, annexin VI may simultaneously mediate an interaction with p120GAP and also an interaction with membrane phospholipids. This may in part explain the mechanism by which p120GAP associates with membranes in response to Ca2+ elevation and suggests the potential importance of annexin VI in the regulation of p21 Ras and the role CaLB domains may play in the specific recognition of cellular membranes.     

Regulation of extracellular signal-regulated kinase activity by p120 RasGAP does not involve its pleckstrin homology or calcium-dependent lipid binding domains but does require these domains to regulate cell proliferation.

The gene encoding for p120 RasGAP, has been disrupted in mice (M. Henkemeyer et al., Nature (Lond.), 377: 695-701, 1995).In this study, using fibroblasts derived from these mouse embryos (Gap-/-; P. van der Geer et al., Mol. Cell Biol., 17: 1840-1847, 1997), we demonstrate that mitogen-activated protein kinase (MAPK) activation is prolonged after epidermal growth factor (EGF), but not lysophosphatidic acid, stimulation as compared with wild-type cells. Furthermore, these cells exhibited a moderate increase in their proliferative rate and saturation density, as well as a limited ability to form colonies in soft agar. Stable cell lines expressing full-length p120GAP not only restored the ability to down-regulate MAPK after EGF stimulation but also lowered their saturation densities. Similarly, expression of p120GAP, missing either its pleckstrin homology (PH) or its calcium-dependent lipid binding (CaLB)/C2 domain, restored MAPK down-regulation and retained the ability to associate with p190 RhoGAP and to be phosphorylated by v-src but exhibited higher saturation densities similar to Gap-/- cells. Our results, therefore, suggest that p120GAP functions not only by down-regulating the Ras/MAPK pathway after growth factor stimulation but is also important in regulating cell proliferation that involves its PH and CaLB domains.     

RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro.

The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.     

P120-Ras GTPase activating protein (RasGAP): A multi-interacting protein in downstream signaling

p120-RasGAP (Ras GTPase activating protein) plays a key role in the regulation of Ras-GTP bound by promoting GTP hydrolysis via its C-terminal catalytic domain. The p120-RasGAP N-terminal part contains two SH2, SH3, PH (pleckstrin homology) and CaLB/C2 (calcium-dependent phospholipid-binding domain) domains. These protein domains allow various functions, such as anti-/pro-apoptosis, proliferation and also cell migration depending of their distinct partners. The p120-RasGAP domain participates in protein–protein interactions with Akt, Aurora or RhoGAP to regulate functions described bellow. Here, we summarize, in angiogenesis and cancer, the various functional roles played by p120-RasGAP domains and their effector partners in downstream signaling.     

The Ras/p120 GTPase-activating protein (GAP) interaction is regulated by the p120 GAP pleckstrin homology domain

Pleckstrin homology domains are structurally conserved functional domains that can undergo both protein/protein and protein/lipid interactions. Pleckstrin homology domains can mediate inter- and intra-molecular binding events to regulate enzyme activity. They occur in numerous proteins including many that interact with Ras superfamily members, such as p120 GAP. The pleckstrin homology domain of p120 GAP is located in the NH(2)-terminal, noncatalytic region of p120 GAP. Overexpression of the noncatalytic domains of p120 GAP may modulate Ras signal transduction pathways. Here, we demonstrate that expression of the isolated pleckstrin homology domain of p120 GAP specifically inhibits Ras-mediated signaling and transformation but not normal cellular growth. Furthermore, we show that the pleckstrin homology domain binds the catalytic domain of p120 GAP and interferes with the Ras/GAP interaction. Thus, we suggest that the pleckstrin homology domain of p120 GAP may specifically regulate the interaction of Ras with p120 GAP via competitive intra-molecular binding.     

Dynamic interaction between Arf GAP and PH domains of ASAP1 in the regulation of GAP activity

ASAP family Arf GAPs induce the hydrolysis of GTP bound to the Ras superfamily protein Arf1, regulate cell adhesion and migration and have been implicated in carcinogenesis. The ASAP proteins have a core catalytic domain of PH, Arf GAP and Ank repeat domains. The PH domain is necessary for both biological and catalytic functions of ASAP1 and has been proposed to be integrally folded with the Arf GAP domain. Protection studies and analytical ultracentrifugation studies previously reported indicated that the domains are, at least partly, folded together. Here, using NMR spectroscopy and biochemical analysis, we have further tested this hypothesis and characterized the interdomain interaction. A comparison of NMR spectra of three recombinant proteins comprised of either the isolated PH domain of ASAP1, the Arf GAP and ankyrin repeat domain or all three domains indicated that the PH domain did interact with the Arf GAP and Ank repeat domains; however, we found a significant amount of dynamic independence between the PH and Arf GAP domains, consistent with the interactions being transient. In contrast, the Arf GAP and Ank repeat domains form a relatively rigid structure. The PH-Arf GAP domain interaction partially occluded the phosphoinositide binding site in the soluble protein, but binding studies indicated the PIP2 binding site was accessible in ASAP1 bound to a lipid bilayer surface. Phosphoinositide binding altered the conformation of the PH domain, but had little effect on the structure of the Arf GAP domain. Mutations in a loop of the PH domain that contacts the Arf GAP domain affected PIP2 binding and the K(m) and k(cat) for converting Arf1 GTP to Arf1 GDP. Based on these results, we generated a homology model of a composite PH/Arf GAP/Ank repeat domain structure. We propose that the PH domain contributes to Arf GAP activity by either binding to or positioning Arf1 GTP that is simultaneously bound to the Arf GAP domain.     

Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P(2) binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.     

Distinct phosphoinositide binding specificity of the GAP1 family proteins: characterization of the pleckstrin homology domains of MRASAL and KIAA0538

GAP1, one of the Ras GTPase-activating protein families, includes four distinct genes (GAP1(m), GAP1(IP4BP), MRASAL (murine Ras GTPase-activating-like), and KIAA0538). It contains an amino-terminal tandem C2 domain, a GAP-related domain, and a carboxyl-terminal pleckstrin homology (PH) domain. Although the PH domains of GAP1(m) and GAP1(IP4BP) have been shown to be essential for membrane targeting via binding of specific phospholipids, little is known about the functions of the PH domains of MRASAL and KIAA0538. Herein, we show that the PH domain of MRASAL has binding activity toward PI(4,5)P(2) and PI(3,4,5)P(3), while the PH domain of KIAA0538 does not bind these phospholipids due to an amino acid substitution at position 592 (Leu-592). Mutation of the corresponding position of MRASAL (Arg-to-Leu substitution at position 591) resulted in loss of the phospholipid binding activity. MRASAL proteins were localized at the plasma membrane in NIH3T3 cells, and this plasma membrane association was unchanged even after cytochalasin B or wortmannin treatment. By contrast, KIAA0538 and MRASAL (R591L) proteins were present in the cytosol. Our data indicate that the distinct phosphoinositide binding specificity of the PH domain is attributable to the distinct subcellular localization of the GAP1 family.     

Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP.     

Identification of residues in GTPase-activating protein Src homology 2 domains that control binding to tyrosine phosphorylated growth factor receptors and p62.

Ras GTPase-activating protein (GAP) contains two Src homology 2 (SH2) domains which are implicated in binding to tyrosine-phosphorylated sites in specific activated growth factor receptors and to a cytoplasmic tyrosine-phosphorylated protein, p62. We have used site-directed mutagenesis of the two GAP SH2 domains (SH2-N and SH2-C) to identify residues involved in receptor and p62 binding. A bacterial fusion protein containing the precise SH2-N domain, as defined by sequence homology, associated with both the activated beta platelet-derived growth factor receptor and epidermal growth factor receptor, and p62 in vitro. However, short deletions at either the N or C termini of the SH2-N domain abolished binding, suggesting that the entire SH2 sequence is required for formation of an active domain. Conservative substitutions of 2 highly conserved basic residues in the SH2-N domain, an arginine and a histidine, resulted in complete loss of receptor and p62 binding, whereas other basic residues, and residues at variable SH2 sites, were more tolerant of substitution. The conserved arginine and histidine therefore appear critical for association with phosphotyrosine-containing proteins, possibly through an interaction with phosphotyrosine. The GAP SH2-C domain, unlike SH2-N, does not bind efficiently to activated receptors or p62 in vitro. The SH2-C domain lacks 3 residues which are otherwise well conserved, and contribute to high affinity SH2-N binding. Replacement of 1 of these residues, a cysteine, with the consensus glycine, conferred SH2-C binding activity toward tyrosine-phosphorylated p62 and epidermal growth factor receptor. Loss-of-function and gain-of-function mutations in the GAP SH2 domains can therefore be used to identify residues that are critical for receptor and p62 binding.     

GTP-binding protein-like domain of AGAP1 is protein binding site that allosterically regulates ArfGAP protein catalytic activity

AGAPs are a subtype of Arf GTPase-activating proteins (GAPs) with 11 members in humans. In addition to the Arf GAP domain, the proteins contain a G-protein-like domain (GLD) with homology to Ras superfamily proteins and a PH domain. AGAPs bind to clathrin adaptors, function in post Golgi membrane traffic, and have been implicated in glioblastoma. The regulation of AGAPs is largely unexplored. Other enzymes containing GTP binding domains are regulated by nucleotide binding. However, nucleotide binding to AGAPs has not been detected. Here, we found that neither nucleotides nor deleting the GLD of AGAP1 affected catalysis, which led us to hypothesize that the GLD is a protein binding site that regulates GAP activity. Two-hybrid screens identified RhoA, Rac1, and Cdc42 as potential binding partners. Coimmunoprecipitation confirmed that AGAP1 and AGAP2 can bind to RhoA. Binding was mediated by the C terminus of RhoA and was independent of nucleotide. RhoA and the C-terminal peptide from RhoA increased GAP activity specifically for the substrate Arf1. In contrast, a C-terminal peptide from Cdc42 neither bound nor activated AGAP1. Based on these results, we propose that AGAPs are allosterically regulated through protein binding to the GLD domain.     

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains

ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.     

Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.     

ArhGAP15, a novel human RacGAP protein with GTPase binding property

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed specificity towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and over-expression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress fibers and cell contraction. These morphological effects can be attenuated by the co-expression of dominant negative Rac1(N17). HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1.     

Dual regulation of RA-RhoGAP activity by phosphatidic acid and Rap1 during neurite outgrowth

During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of a novel Rho GTPase activating protein implicated in receptor-mediated endocytosis

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing Rho1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.     

Molecular characterization of protein kinase C-alpha binding to lamin A

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of rat PKC-alpha, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200-217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC-alpha to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca(2+). We have also found that the binding site of lamin A for the CaLB domain of PKC-alpha is localized in the carboxyl-terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme-specific drugs to attenuate or reverse PKC-dependent nuclear signaling pathways important for the pathogenesis of cancer.     

Pleckstrin homology domains of tec family protein kinases.

Pleckstrin homology (PH) domains have been shown to be involved in different interactions, including binding to inositol compounds, protein kinase C isoforms, and heterotrimeric G proteins. In some cases, the most important function of PH domains is transient localisation of proteins to membranes, where they can interact with their partners. Tec family protein tyrosine kinases contain a PH domain. In Btk, also PH domain mutations lead into an immunodeficiency, X-linked agammaglobulinemia (XLA). A new disease-causing mutation was identified in the PH domain. The structures for the PH domains of Bmx, Itk, and Tec were modelled based on Btk structure. The domains seem to have similar scaffolding and electrostatic polarisation but to have some differences in the binding regions. The models provide new insight into the specificity, function, and regulation of Tec family kinases.