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M Rahmatullah

Publications and source records attributed to M Rahmatullah.

At least 19 recordsLinked to original sources
PubMed1998

Synergistic regulation of Schwann cell proliferation by heregulin and forskolin.

A peptide corresponding to the epidermal growth factor homology domain of beta-heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95(RSK2), was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.

Amino Acid Sequence↗
PubMed1997

Differential coupling of alpha1-adrenoreceptor subtypes to phospholipase C and mitogen activated protein kinase in neonatal rat cardiac myocytes.

Activation of cardiac alpha1-adrenoreceptors has a number of physiological effects. Ascribing these effects to a specific alpha1-adrenoreceptor subtype first requires the elucidation of the subtypes that are present in the tissue of interest. In the present study, mRNA transcripts for the alpha1A, alpha1B and alpha1D-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using reverse transcriptase-polymerase chain reaction analysis. However, binding sites for only the alpha1A and alpha1B-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using competition binding analysis with a variety of alpha1 selective receptor antagonists. Phenylephrine-stimulated phosphatidylinositol hydrolysis was inhibited by alpha1 selective receptor antagonists with affinities consistent with the alpha1A-adrenoreceptor subtype, whereas phenylephrine-induced activation of the mitogen activated protein kinase cascade was inhibited by these same antagonists with affinities more closely resembling the alpha1B-adrenoreceptor subtype. In the case of both signaling pathways, the alpha1D selective receptor antagonist, BMY 7378, exhibited affinities suggestive of the relative absence of a alpha1D-adrenoreceptor subtype. Thus, despite the presence of mRNA transcripts for all three alpha1-adrenoreceptor subtypes, only the alpha1A and alpha1B-adrenoreceptor subtypes were expressed and functionally coupled at detectable levels in neonatal rat cardiac myocytes. Of particular interest, phenylephrine-induced activation of the mitogen activated protein kinase cascade appears to be mediated by a subtype resembling most closely the pharmacological profile of the alpha1B-adrenoreceptor subtype.

Animals↗
PubMed1995

Specificity of G protein alpha-gamma subunit interactions. N-terminal 15 amino acids of gamma subunit specifies interaction with alpha subunit.

The existence of multiple alpha, beta, and gamma subunits raises questions regarding the assembly of particular G proteins. Based on the results of a previous study (Rahmatullah, M., and Robishaw, J. D. (1994) J. Biol. Chem. 269, 3574-3580), we hypothesized that the assembly of G proteins may be determined by the interactions of the more structurally diverse alpha and gamma subunits. This hypothesis was confirmed in the present study by showing striking differences in the abilities of the gamma 1 and gamma 2 subunits to interact with the alpha o subunit. Chimeras of the gamma 1 and gamma 2 subunits were used to delineate which region is responsible. Support for the importance of the N-terminal region of the gamma subunit comes from our observations that 1) the gamma 2 subunit and the gamma 211 chimera bound strongly to the alpha o-agarose matrix, but the gamma 1 subunit and the gamma 112 chimera bound weakly, if at all; 2) an N-terminal peptide made to the gamma 2 subunit blocked the binding of the gamma 211 chimera to the alpha o-agarose matrix; 3) both the gamma 211 chimera and the N-terminal peptide were able to partially protect the alpha o subunit against tryptic cleavage; and 4) the gamma 211 chimera, but not the gamma 112 chimera, supported ADP-ribosylation of the alpha o subunit.

Adenosine Diphosphate Ribose↗
PubMed1994

Direct interaction of the alpha and gamma subunits of the G proteins. Purification and analysis by limited proteolysis.

The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.

Animals↗
PubMed1993

Partial activation of the pyruvate dehydrogenase kinase by the lipoyl domain region of E2 and interchange of the kinase between lipoyl domain regions.

The binding of the pyruvate dehydrogenase (E1) component and the E1-specific kinase to the core-forming dihydrolipoyl acetyltransferase (E2) component facilitates a severalfold enhancement in the rate at which the kinase phosphorylate E1 (i.e. versus free kinase phosphorylating free E1). The kinase and E1 associate with small exterior linker region-connected domains in the E2 structure. The kinase binds to one of two lipoyl domains, and the E1 component binds to a domain in E2"s structure between the lipoyl domain region and the inner domain. Sixty of the latter domains assemble to form a dodecahedron-shaped inner core. Binding of the kinase to a detached lipoyl domain region enhanced kinase activity. This bi-lipoyl domain fragment induced a 2-fold enhancement in the slow rate of phosphorylation of peptide substrate and intact E260 gave only a 50% higher rate. In contrast, the lipoyl domain fragment gave only a 40% enhancement in the faster rate of phosphorylation of E1; whereas the rate of phosphorylation of E1 was markedly increased (4-10-fold depending on conditions) by kinase interaction with the intact E2 core. Binding of E1 to an E2 structure lacking only the bi-lipoyl domain region did not enhance kinase activity. Thus, binding of the kinase to the lipoyl domain region elicits a structural change which enhances kinase activity; however, other processes are required to explain the very large enhancement in phosphorylation of E1 effected by intact E2 core. Among the latter is a need for a mechanism allowing one kinase molecule to phosphorylate many E1 tetramers, whereas both E1 and the kinase stay bound to the oligomeric E2 core (i.e. phosphorylation appears to be much faster than the dissociation of either the kinase or E1 tetramers from E260 core). Exposure of kinase bound to the lipoyl domain fragment to intact E2 core for 10 s allowed a transition to a maximal (7-fold) activation of the kinase. In the opposite direction, an increasing level of the free bi-lipoyl domain fragment rapidly reduced, in a concentration-dependent manner the activity of kinase bound initially to intact E2. The data strongly support kinase transfer between free lipoyl domains and the intact E2 core and fit about a 12-fold tighter binding of the kinase to intact E2 cores over binding to free lipoyl domains. Such an interchange of the kinase between these E2 structures was confirmed by sucrose gradient studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed1991

Characterization of a cAMP-independent Ca2(+)-inhibited protamine kinase from Candida lipolytica.

A cAMP-independent protamine kinase has been purified from extracts of the yeast Candida lipolytica by ion-exchange and affinity chromatography. Two subunits with apparent Mr's of 52,000 and 36,000 were resolved by SDS-PAGE. The purified kinase exhibited about 20% activity with casein and histone Type VII-S as substrates relative to protamine. The enzyme was inactive against other protein substrates tested, and was essentially insensitive to AMP, cAMP, cGMP up to 0.2 mM, the polyamines spermine and spermidine up to 1 mM, N-ethylmaleimide (5 mM), 2-mercaptoethanol (20 mM), or dithiothreitol (2 mM), and several cations like Zn2+, N1+, or Co2+ at 0.1 mM each. Ca2+ at 3 mM inhibited protamine kinase activity by 50%, which was reversed by EGTA.

Calcium↗
PubMed1990

Changes in the core of the mammalian-pyruvate dehydrogenase complex upon selective removal of the lipoyl domain from the transacetylase component but not from the protein X component.

The dihydrolipoyl transacetylase (E2) component contains a COOH-terminal inner domain (E2I) and an extended NH2-terminal structure, which is composed of two lipoyl domains (the fragment containing both is designated as E2L) and a subunit-binding domain (E2B). The four domains are connected by hinge regions. A subcomplex, composed of an oligomer of E2 subunits, protein X (which also has an NH2-terminal lipoyl domain), and the [pyruvate dehydrogenase]-kinase catalytic and basic subunits (Kc and Kb, respectively) (i.e. E2.X.KcKb subcomplex), was treated with Clostridium histolyticum collagenase. E2 subunits were selectively cleaved at the NH2-terminal end of the E2B domain, releasing the E2L fragment. Complete release of E2 subunits also released the kinase subunits, indicating that the kinase is bound to the E2L portion of E2. The residual inner core subcomplex (designated E2IB.X) has a strong tendency to aggregate, but this can be reversed with heparin (1 mg/ml). The E2IB.X subcomplex binds the pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components. The E1 component, which binds to the E2B domain, blocked collagenase cleavage of E2. We evaluated the capacity of the collagenase-treated E2.X.KcKb subcomplex, from which different portions of the E2L domains were removed, to support (in combination with excess levels of the E1 and E3 components) the overall reaction of the complex. Loss of activity occurred only after more than half of the E2L domains were removed. This delay is in sharp contrast to the effect of selective removal of the lipoyl domain of protein X, which leads to an immediate decrease in activity (Gopalakrishnan, S., Rahmatullah, M., Radke, G.-A., Powers-Greenwood, S. L., and Roche, T. E. (1989) Biochem. Biophys. Res. Commun. 160, 715-721). These results suggest that multiple lipoyl domains of the E2 component service the rate-limiting E1 component. After all the E2L domains were removed and the E2IB.X subcomplex was separated from free E2L, 10% activity was retained in the overall reaction. Thus, the lipoyl domain of protein X supported the overall reaction of the complex.

Acetyltransferases↗
PubMed1989

Role of protein X in the function of the mammalian pyruvate dehydrogenase complex.

Two lipoyl-bearing subunits--the dihydrolipoyl transacetylase and protein X--form the core of the mammalian pyruvate dehydrogenase complex. Selective removal of the lipoyl domain of protein X results in loss in the activity of the complex with a relationship suggesting the involvement of the lipoyl domain of protein X in a key but not rate limiting step. The dihydrolipoyl dehydrogenase component markedly reduces both the cleavage of protein X and the loss in activity. Using a microplate binding assay, we demonstrate that the lipoyl domain of protein X and the transacetylase component contribute to the binding of the dihydrolipoyl dehydrogenase component. These roles of protein X in the catalytic function and organization of the complex require new reactions and afford an explanation for the unusual stoichiometry of dihydrolipoyl dehydrogenase dimers in the complex.

Animals↗
PubMed1989

Separation of protein X from the dihydrolipoyl transacetylase component of the mammalian pyruvate dehydrogenase complex and function of protein X.

The dihydrolipoyl transacetylase (E2)-protein X-kinase subcomplex was resolved to produce an oligomeric transacetylase that was free of protein X and kinase subunits. We investigated the properties of this transacetylase E2 oligomer and of a form of the subcomplex from which only the lipoyl-bearing domain of protein X (XL) was removed. While retaining other catalytic and binding properties of the native subcomplex, the oligomeric transacetylase and the subcomplex lacking the XL domain had greatly reduced capacities both to support the overall reaction of the complex (upon reconstitution with other components) and to bind the dihydrolipoyl dehydrogenase component. Our results indicate that protein X, in part through its XL domain, contributes to the binding of the dihydrolipoyl dehydrogenase component and to the overall reaction of the complex.

Acetyltransferases↗
PubMed1989

Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component.

We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.

Acetyltransferases↗
PubMed1989

Domain structures of the dihydrolipoyl transacetylase and the protein X components of mammalian pyruvate dehydrogenase complex. Selective cleavage by protease Arg C.

Treatment of the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2-X-KcKb) with protease Arg C selectively converted protein X into an inner domain fragment (Mr approximately equal to 35,000) and an outer (lipoyl-bearing) domain fragment (Mr approximately equal to 15,500). These fragments were larger and much smaller, respectively, than the inner domain and outer domain fragments derived from the E2 component, supporting the conclusion that protein X is distinct from the E2 component. Protease Arg C cleaved the Kb subunit more slowly than protein X. An increase in kinase activity correlated with this cleavage of the Kb subunits. An even slower cleavage of E2 subunits generated an inner domain fragment (Mr approximately equal to 31,500) and a lipoyl-bearing domain fragment (Mr approximately equal to 49,000) which had Mr values at least 3,000 and 10,000 larger, respectively, than the corresponding E2 fragments generated by trypsin treatment of the subcomplex. Following various extents of cleavage with protease Arg C or trypsin, residual oligomeric subcomplexes were isolated and characterized. We found that selective removal of the lipoyl-bearing domain of protein X did not alter lipoyl-mediated regulation of the kinase indicating that the lipoyl residues bound to E2 subunits are effective, that the inner domain of protein X remained associated with the inner domain of E2 subunits following the complete removal of the outer domains of both E2 and protein X, that, with only 10% of the E2 subunits intact, nearly half of the catalytic (Kc) subunits of the kinase were bound by the residual subcomplex, and that removal of the remaining outer domains from E2 subunits released the Kc subunits. Thus, protein X is unique among the subunits of the complex in binding tightly to the oligomeric inner domain of the transacetylase, and the outer domain of the transacetylase serves to bind to and facilitate the regulation of the catalytic subunit of the kinase.

Acyltransferases↗
PubMed1988

Component requirements for NADH inhibition and spermine stimulation of pyruvate dehydrogenaseb phosphatase activity.

The subunit and subdomain requirements for NADH inhibition as well as Ca+ and spermine activation of the pyruvate dehydrogenaseb phosphatase were analyzed. The transacetylase-protein X subcomplex (E2-X) was required for all three effects. The oligomeric inner domain of the transacetylase did not support any of these regulatory effects. The presence of at least a portion of the outer (lipoyl-bearing) domains of the transacetylase but not the lipoyl-bearing portion of protein X was essential for expression of these regulatory effects on phosphatase activity. The inner domain of protein X may contribute to some effects. The E2-X subcomplex, alone, had no effect on phosphatase activity in the absence of Ca2+, but the subcomplex did support both NADH inhibition and spermine activation in the absence of Ca2+. Studies with peptide substrates established that spermine is directly bound by a phosphatase subunit. With the resolved pyruvate dehydrogenase component (E1b) used as the substrate, the E2-X subcomplex transformed the effect of spermine from inhibiting to stimulating the rate of dephosphorylation by the phosphatase. The above observations suggest that binding of E1b to the E2-X subcomplex alters its presentation to the phosphatase. We also present several observations that are consistent with NADH inhibition of the phosphatase being mediated through a dihydrolipoyl dehydrogenase-dependent reduction of lipoyl moieties in the E2-X subcomplex. Overall, our data establish that the outer, lipoyl-bearing domains of the oligomeric transacetylase core have an essential role in the function and regulation of the pyruvate dehydrogenase phosphatase.

Alkylation↗
PubMed1987

The catalytic requirements for reduction and acetylation of protein X and the related regulation of various forms of resolved pyruvate dehydrogenase kinase.

The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.

Acetylation↗
PubMed1986

Properties of the pyruvate dehydrogenase kinase bound to and separated from the dihydrolipoyl transacetylase-protein X subcomplex and evidence for binding of the kinase to protein X.

Studies were conducted on four pyruvate dehydrogenase kinase-containing fractions: purified pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2.X.K), a kinase fraction (K fraction) prepared from the E2.X.K subcomplex, and a kinase fraction generated by limited trypsin-digestion of E2.X.K. We characterized the gel electrophoresis properties of dissociated subunits (one-dimensional and two-dimensional), the catalytic and ATP binding properties of kinase-containing fractions, and the subunit requirements for kinase binding to and being activated by the transacetylase-protein X subcomplex (E2.X). A significant portion of protein X was retained with the transacetylase core following release of virtually all the kinase. The K fraction had four major bands separated by sodium dodecyl sulfate-slab gel electrophoresis which corresponded to the dihydrolipoyl dehydrogenase, protein X, the trypsin-resistant catalytic subunit of the kinase and a chymotrypsin-resistant subunit which had a high pI and comigrated in one-dimensional systems with the chymotrypsin-sensitive alpha-subunit of the pyruvate dehydrogenase component. While purified kidney complex contained only about three molecules of kinase (determined by [14C]ATP binding), one molecule of E2.X subcomplex activated a large number (greater than 15) molecules of kinase associated with the protein X-containing K fraction. Sephadex G-200 chromatography of the K fraction in the presence of dithiothreitol led to coelution of protein X and kinase subunits. Limited trypsin digestion converted the transacetylase into subdomains and cleaved protein X and the high pI subunit of the kinase. Under those conditions, the intact catalytic subunit of the kinase did not bind to the large inner domain of the transacetylase but could be activated by untreated E2.X subcomplex. Thus, binding of the catalytic subunit of the kinase and its activation by E2.X required either protein X or the lipoyl-bearing outer domain of the transacetylase. In combination, our results suggest that protein X serves to anchor the kinase to the core of the complex.

Acetyltransferases↗
PubMed1986

Properties of a newly characterized protein of the bovine kidney pyruvate dehydrogenase complex.

The dihydrolipoyl transacetylase component, which serves as the structural core of mammalian pyruvate dehydrogenase complexes, is acetylated when treated with either pyruvate or with acetyl-CoA in the presence of NADH. Besides the dihydrolipoyl transacetylase component, we have found that another protein, referred to as protein X, is rapidly acetylated at thiol residues. Protein X remains fully bound to the transacetylase core under conditions that remove the pyruvate dehydrogenase and dihydrolipoyl dehydrogenase components. Mapping of 125I-tryptic peptides indicated that the transacetylase subunits and protein X are structurally distinct; however, under the same mapping conditions, there is considerable similarity in the positions of acetylated peptides derived from these subunits. Affinity-purified rabbit immunoglobulin G prepared against the dihydrolipoyl transacetylase core reacted exclusively with the transacetylase and with both its tryptic-derived inner domain and outer lipolyl-bearing domain. Those results further indicate that protein X is not derived from the transacetylase subunit Affinity-purified mouse antibody to protein X reacted selectively with large tryptic polypeptides derived from protein X and did not react with the inner domain of the transacetylase. However, the anti-protein X antibody did react with the intact transacetylase subunit, the lipoyl-bearing domain of the transacetylase, and weakly with the transsuccinylase component of the alpha-ketoglutarate dehydrogenase complex. This cross-reactivity reflected specificity of a portion of the polyclonal antibodies for a related structural region in the transacetylase and protein X (possibly a similar lipoyl-bearing region). Furthermore, a major portion of that polyclonal antibody was shown to react exclusively with protein X. Thus, protein X subunits differ substantially from transacetylase subunits but the two components have a region of structural similarity. We estimate that there are about 5 mol of protein X per mol of the kidney pyruvate dehydrogenase complex. Under a variety of conditions that result in a wide range of levels of acetylation of sites in the complex, about 1 acetyl group is incorporated into protein X per 10 acetyl groups incorporated into the transacetylase subunits per mol of complex. That ratio is close to the ratio of protein X subunits of transacetylase subunits in the complex, indicating that there are efficient mechanisms for acylation and deacylation of protein X.

Acetylation↗