Biomedical subjects

C Smythe

Publications and source records attributed to C Smythe.

At least 19 recordsLinked to original sources

PubMed2001

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing.

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

Alkaloids↗

PubMed2000

INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.

Amino Acid Sequence↗

PubMed2000

Assembly of U7 small nuclear ribonucleoprotein particle and histone RNA 3' processing in Xenopus egg extracts.

In animals, replication-dependent histone genes are expressed in dividing somatic cells during S phase to maintain chromatin condensation. Histone mRNA 3'-end formation is an essential regulatory step producing an mRNA with a hairpin structure at the 3'-end. This requires the interaction of the U7 small nuclear ribonucleoprotein particle (snRNP) with a purine-rich spacer element and of the hairpin-binding protein with the hairpin element, respectively, in the 3'-untranslated region of histone RNA. Here, we demonstrate that bona fide histone RNA 3' processing takes place in Xenopus egg extracts in a reaction dependent on the addition of synthetic U7 RNA that is assembled into a ribonucleoprotein particle by protein components available in the extract. In addition to reconstituted U7 snRNP, Xenopus hairpin-binding protein SLBP1 is necessary for efficient processing. Histone RNA 3' processing is not affected by addition of non-destructible cyclin B, which drives the egg extract into M phase, but SLBP1 is phosphorylated in this extract. SPH-1, the Xenopus homologue of human p80-coilin found in coiled bodies, is associated with U7 snRNPs. However, this does not depend on the U7 RNA being able to process histone RNA and also occurs with U1 snRNPs; therefore, association of SPH1 cannot be considered as a hallmark of a functional U7 snRNP.

3' Untranslated Regions↗

PubMed2000

Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs.

In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B(3) specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B(3) co-immunoprecipitate, and lamin B(3) interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB delta 2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.

Amino Acid Sequence↗

PubMed1999

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK.

ATR is a large, > 300 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with the DNA-PK, are part of a new family of PI-3 kinase related proteins. All members of this family play important roles in checkpoints which operate to permit cell survival following many forms of DNA damage. We have expressed ATR protein in HEK293 cells and purified the protein to near-homogeneity. We show that pure ATR is a protein kinase which is activated by circular single-stranded, double-stranded or linear DNA. Thus ATR is a new member of a sub-family of PIK related kinases, founded by the DNA-PK, which are activated in the presence of DNA. Unlike DNA-PK, ATR does not appear to require Ku proteins for its activation by DNA. We show directly that, like ATM and DNA-PK, ATR phosphorylates the genome surveillance protein p53 on serine 15, a site which is up-regulated in response to DNA damage. In addition, we find that ATR has a substrate specificity similar to, but unique from, the DNA-PK in vitro, suggesting that these proteins have overlapping but distinct functions in vivo. Finally, we find that the kinase activity of ATR in the presence and absence of DNA is suppressed by caffeine, a compound which is known to induce loss of checkpoint control. Our results are consistent with the notion that ATR plays a role in monitoring DNA structure and phosphorylation of proteins involved in the DNA damage response pathways.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1999

Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types.

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

Animals↗

PubMed1998

PD 98059 prevents establishment of the spindle assembly checkpoint and inhibits the G2-M transition in meiotic but not mitotic cell cycles in Xenopus.

Most chemotherapeutic agents block DNA replication, damage DNA, or interfere with chromosome segregation. The existence of checkpoints, which monitor these events, indicates that mechanisms exist to avoid death when essential cellular events are inhibited. A molecular understanding of cellular checkpoints should therefore provide opportunities for the development of inhibitors of checkpoint controls which may increase the potency of chemotherapeutic drugs by inducing catastrophic cell cycle progression. The molecular dissection of cell cycle arrest points is facilitated in the Xenopus egg/oocyte system, in which cell-free systems retain both S/M and spindle assembly checkpoints. Members of the MAP kinase family have been shown to play a role in the induction of G2 to M transition during oocyte maturation and have been implicated in the maintenance of either cytostatic factor- or spindle assembly checkpoint-induced M-phase arrest. Here, we have examined the effects of the inhibitor of MAP kinase kinase activation, PD 98059, on cell cycle progression in Xenopus oocytes and in cell-free extracts. This inhibitor is highly specific for the kinase which activates the classical p42/p44 MAP kinase, having no effect on upstream activators of stress-activated protein kinases. We have found that PD 98059 inhibits oocyte maturation, consistent with a role for p42 MAP kinase as a rate-limiting component in the induction of meiosis, but had no effect on the timing of G2-M transition in cell-free extracts indicating that, unlike meiosis, p42 MAP kinase activation is not limiting for normal mitotic M phase entry. However, we found that cytostatic factor-induced metaphase arrest, as well as the spindle assembly checkpoint, were both abolished in the presence of the drug. These results demonstrate that p42 MAP kinase, and not some other member of the MAP kinase family, is responsible for both CSF- and checkpoint-induced metaphase arrest and suggest that PD 98059 and similar agents may have considerable therapeutic potential for the potentiation of chemotherapeutic regimes.

Animals↗

PubMed1997

Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1.

BACKGROUND: A common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21(WAF1) protein appears to be the inhibition of G1 cyclin-Cdk complexes. Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative drugs for use in tumours with a defective p53 pathway. RESULTS: We report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21(WAF1) inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21(WAF1) in the inhibition of Cdk4 activity. CONCLUSIONS: We have discovered that a small peptide is sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.

Amino Acid Sequence↗

PubMed1996

Vacuum erection device in spinal cord injured men: patient and partner satisfaction.

OBJECTIVE: To assess the efficacy of and patient and partner satisfaction with a vacuum erection device (VED) to treat erectile dysfunction of spinal cord injury. DESIGN: Case series. SETTING: University hospital outpatient clinic. PATIENTS: Twenty spinal cord injured men with erectile dysfunction and their heterosexual partners, recruited from outpatient population and by advertisement. INTERVENTION: Use of a VED to obtain erections for sexual activity. MAIN OUTCOME MEASURES: Efficacy in obtaining adequate penile erection, and patient and partner satisfaction with the device (survey). RESULTS: At 3 months, 93% of the men and 83% of the women reported rigidity sufficient for vaginal penetration, with an average duration of 18 minutes. These numbers decreased somewhat at the 6-month control. At 6 months, 41% of the men and 45% of the women were satisfied with the device, with premature loss of rigidity during intercourse the most commonly reported complaint. Sixty percent of men and 42% of women indicated an improvement of the sexual relationship. Minor side effects, such as petechiae and penile skin edema, occurred frequently, but there were no complications that required treatment. CONCLUSION: The VED is effective in many couples in the treatment of erectile dysfunction associated with spinal cord injury. The devices were not universally accepted, but had a significant impact on sexual activity and sexual satisfaction for nearly half the couples. Vacuum erection devices should be presented to SCI men along with other options for treatment of erectile dysfunction.

Adult↗

PubMed1996

Activation of the Xenopus cyclin degradation machinery by full-length cyclin A.

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin. A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.

Animals↗

PubMed1995

Isolation and characterisation of a uniquely regulated threonine, tyrosine phosphatase (TYP 1) which inactivates ERK2 and p54jnk.

The recent discovery of the vaccinia virus protein phosphatase VH1, and its mammalian counterparts has highlighted a novel subfamily of protein tyrosine phosphatases that exhibit dual specificity toward phosphotyrosine- and phosphoserine/threonine-residues. We have identified further members of this subfamily. The characterisation of one clone in particular, which we have named threonine-tyrosine phosphatase 1 (TYP 1), encodes a protein homologous to CL100, but differs dramatically in its regulation. TYP 1 is not expressed in human fibroblasts unlike other CL100-like genes. Furthermore, northern analysis has demonstrated that following mitogenic stimulation of squamous cells, induction of TYP 1 mRNA reaches its maximal levels after four hours, in contrast to the immediate early CL100-like genes. Both TYP 1 and CL100 mRNAs are induced upon TGF-beta treatment of squamous cell lines sensitive to the growth factors antiproliferative effects. When TYP 1 is transfected into COS-1 cells, the gene product inhibits both ERK2 and p54 MAP kinase subfamilies. In addition, we show that purified TYP 1 protein efficiently inactivates recombinant ERK2 in vitro by the concomitant dephosphorylation of both its phosphothreonine and -tyrosine residues. TYP 1 encodes a nuclear protein, which when expressed in COS cells is stabilised by EGF treatment.

3T3 Cells↗

PubMed1995

XCL100, an inducible nuclear MAP kinase phosphatase from Xenopus laevis: its role in MAP kinase inactivation in differentiated cells and its expression during early development.

We have cloned the Xenopus laevis homologue (XCL100) of the human CL100 (Thr/Tyr) MAP kinase phosphatase. Expression of the XCL100 mRNA and protein is inducible by serum stimulation and oxidative/heat stress in a X. laevis kidney cell line. In contrast, XCL100 is constitutively expressed in growing Xenopus oocytes. Recombinant XCL100 protein is able to dephosphorylate both tyrosine and threonine residues of activated p42 MAP kinase in vitro and both the Xenopus and human CL100 proteins were localised predominantly in the nucleus in transfected COS-1 cells. As nuclear translocation of activated MAP kinase is necessary for some of its essential functions in proliferation and cell differentiation our results indicate a role for CL100 in the regulation of these nuclear signalling events. In Xenopus kidney cells both heat shock and serum stimulation lead to transient activation of MAP kinase. However, in contrast to results previously reported from studies on mammalian fibroblasts the inactivation of MAP kinase in these epitheloid cells is rapid and is not dependent on synthesis of new protein. These results indicate that the induction of CL100 (or CL100-like enzymes) may not be required for MAP kinase inactivation in all cell types. Finally, during early embryogenesis, levels of XCL100 mRNA are greatly increased at the mid-blastula transition, suggesting that this enzyme may be involved in the regulation of MAP kinase activity during early development.

Amino Acid Sequence↗

PubMed1995

An analysis of specialty journals on alcohol, drugs and addictive behaviors for sex bias in research methods and reporting.

OBJECTIVE: Several studies have considered the extent to which gender bias has characterized addictions treatment research over the past 20 years. Sex bias in this literature has been shown to be most apparent in a reliance on male subjects and representation of male experience as normative. The current study was undertaken to assess the current status of some basic aspects of gender bias in addictions research generally. METHODS: Articles appearing in 1990 in 17 specialty journals on alcohol, drugs and addictive behaviors were coded with respect to type of article and the gender of the primary investigator. Studies on human subjects also were evaluated with regard to subject selection and reporting practices. RESULTS: Although the proportion of females represented in addictions research was found to have increased over earlier historical periods, studies using only male subjects were still common in 1990. Moreover, in studies using subjects of mostly one sex, investigators were likely to provide legitimate reasons for studying females, but provide no rationale for using male samples. Other subtle forms of bias were identified. For example, many single-sex studies (especially those using all male subjects) did not indicate the gender of the sample in the title or abstract of the study, and did not indicate that results of the study could be generalized to only one gender. CONCLUSIONS: Male biased sampling and misleading reporting of findings continue to be evident in addictions research. The need for gender-sensitive research in the field of addictions is discussed, and suggestions for change are offered.

Alcoholism↗

PubMed1993

The human CL100 gene encodes a Tyr/Thr-protein phosphatase which potently and specifically inactivates MAP kinase and suppresses its activation by oncogenic ras in Xenopus oocyte extracts.

The expression of the human CL100 gene and its mouse homologue 3CH134 is increased up to 40-fold in fibroblasts exposed to oxidative/heat stress and growth factors. CL100 is a member of an expanding family of protein tyrosine phosphatases with amino acid sequence similarity to a Tyr/Ser-protein phosphatase encoded by the late H1 gene of vaccinia virus. Here we show that the CL100 phosphatase, expressed and purified in bacteria, rapidly and potently inactivates recombinant MAP kinase in vitro by the concomitant dephosphorylation of both its phosphothreonine and phosphotyrosine residues. Furthermore, CL100 suppresses the [val12] ras-induced activation of MAP kinase in a cell-free system from Xenopus oocytes. Both activities are abolished by mutagenesis of the highly conserved cysteine (Cys-258) within the phosphatase active site. In contrast to the vaccinia H1 phosphatase, CL100 shows no measurable catalytic activity towards a number of other substrate proteins modified on serine, threonine or tyrosine residues. Our results demonstrate that CL100 is a dual specificity phosphatase and indicate that MAP kinase is one of its physiological targets. CL100 may be the first example of a new class of protein phosphatases responsible for modulating the activation of MAP kinase following exposure of quiescent cells to growth factors and further implicates MAP kinase activation/deactivation in the cellular response to stress.

Amino Acid Sequence↗

PubMed1992

Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2.

In cell-free extracts derived from Xenopus eggs which oscillate between S phase and mitosis, incompletely replicated DNA blocks the activation of p34cdc2-cyclin by maintaining p34cdc2 in a tyrosine-phosphorylated form. We used a recombinant cyclin fusion protein to generate a substrate to measure the ability of the tyrosine kinase(s) to phosphorylate and inactivate p34cdc2 in the absence of tyrosine phosphatase activity. p34cdc2 tyrosine phosphorylation is highly regulated during the cell cycle, being elevated in S phase and attenuated in mitosis. The elevation in p34cdc2 tyrosine phosphorylation rate occurs in response to the presence of incompletely replicated DNA. Moreover, okadaic acid and caffeine, which uncouple the dependence of mitosis on the completion of S phase, increase unphosphorylated p34cdc2 by attenuating tyrosine kinase function. These data indicate that the control system, which monitors the state of DNA replication, modulates the function of the tyrosine kinase by a phosphorylation/dephosphorylation mechanism, ensuring that mitosis occurs only when S phase is complete.

Animals↗

PubMed1992

DNA replication and progression through the cell cycle.

Somatic cells possess control mechanisms which monitor DNA replication and assure that it is complete before mitosis is initiated. We have been investigating these mechanisms in Xenopus egg extracts. Using in vitro cycling extracts, which spontaneously alternate between interphase and mitosis, we found that the onset of mitosis is inhibited by the presence of unreplicated DNA, demonstrating that the completion of DNA replication and the initiation of mitosis are coupled in these extracts. As in somatic cells, this coupling is sensitive to caffeine and to okadaic acid. In Xenopus extracts unreplicated DNA increases the tyrosine phosphorylation of p34cdc2, thereby maintaining MPF (mitosis-promoting factor) in an inactive state and preventing the onset of mitosis. The block to mitosis in the presence of unreplicated DNA can be reversed by the addition of bacterially expressed cdc25 protein. The extent of MPF activation by cdc25 protein under these conditions depends on the number of nuclei present. We have developed an assay to examine the rate of tyrosine phosphorylation on p34cdc2. It is increased by unreplicated DNA, in a manner consistent with unreplicated DNA up-regulating the kinase that phosphorylates p34cdc2. We have begun to examine how unreplicated DNA generates the signal that inhibits MPF activation by testing the ability of naked single- and double-stranded DNA templates to inhibit mitosis, and by investigating the role of RCC1, a chromatin-associated protein required for the coupling of DNA replication and mitosis.

Animals↗

PubMed1992

In vitro cell cycle arrest induced by using artificial DNA templates.

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.

Animals↗

PubMed1991

The discovery of glycogenin and the priming mechanism for glycogen biogenesis.

The biogenesis of glycogen in skeletal muscle requires a priming mechanism that has recently been elucidated. The first step is catalysed by a protein tyrosine glucosyltransferase and involves the formation of a novel glycosidic linkage, namely the covalent attachment of glucose to a single tyrosine residue (Tyr194) on a priming protein, termed glycogenin. The next stage is the extension of the glucan chain from Tyr194 and involves the sequential addition of up to seven further glucosyl residues. This reaction is brought about autocatalytically by glycogenin itself, which is a Mn2+/Mg(2+)-dependent UDP-Glc-requiring glucosyltransferase. The glucan primer is elongated by glycogen synthase, but only when glycogenin and glycogen synthase are complexed together. Glycogen synthase dissociates from glycogenin during the synthesis of a glycogen molecule, enabling glycogen molecules to reach their maximum theoretical size. Each mature glycogen beta particle in muscle contains one molecule of glycogenin attached covalently, and an average one glycogen synthase catalytic subunit bound non-covalently. As evidence accumulates that a priming protein may be a fundamental property of polysaccharide synthesis in general, the molecular details of mammalian glycogen biogenesis may serve as a useful model for other systems.

Amino Acid Sequence↗