Lamar Seibenhener - Academia.edu (original) (raw)

Papers by Lamar Seibenhener

Journal of Neurology & Neurophysiology, 2015

Annual Review of Neuroscience, 2002

▪ Activity-dependent changes in synaptic function are believed to underlie the formation of mem... more ▪ Activity-dependent changes in synaptic function are believed to underlie the formation of memories. Two prominent examples are long-term potentiation (LTP) and long-term depression (LTD), whose mechanisms have been the subject of considerable scrutiny over the past few decades. Here we review the growing literature that supports a critical role for AMPA receptor trafficking in LTP and LTD, focusing on the roles proposed for specific AMPA receptor subunits and their interacting proteins. While much work remains to understand the molecular basis for synaptic plasticity, recent results on AMPA receptor trafficking provide a clear conceptual framework for future studies.

Molecular Cell Biology Research Communications, 1999

Atypical protein kinase C-zeta (PKC-zeta) participates in nerve growth factor (NGF) signaling and... more Atypical protein kinase C-zeta (PKC-zeta) participates in nerve growth factor (NGF) signaling and is required for NGF-induced differentiation of PC12 cells. The biological activity of PKC-zeta is likely mediated by interaction of PKC-zeta with specific proteins. Affinity column chromatography employing the PKC-zeta regulatory domain coupled to glutathione-agarose was used to search for proteins that bound PKC-zeta. Two proteins (59/60 kDa) were recovered from NGF-stimulated PC12 cell lysates that bound the matrix. Western blot analysis of pooled column fractions identified these proteins as tubulin and src, respectively. Using purified preparations of src and tubulin, PKC-zeta was shown to interact with both proteins using blot overlay. To demonstrate a functional interaction in vivo, PC12 cells expressing a temperature-sensitive v-src were shifted to the permissive temperature (37 degrees C), followed by immunoprecipitation. At the permissive temperature where src was active, PKC-zeta was tyrosine phosphorylated and coassociated with src in vivo; by comparison, at the nonpermissive temperature (40 degrees C) PKC-zeta was not tyrosine phosphorylated. Taken together, these findings support a novel role for the interaction of src and atypical PKC in vivo, which is dependent upon the activity of src and the tyrosine phosphorylation state of PKC-zeta.

Journal of cell science, Jan 15, 2014

The dynein motor protein complex is required for retrograde transport of vesicular cargo and for ... more The dynein motor protein complex is required for retrograde transport of vesicular cargo and for transport of aggregated proteins along microtubules for processing and degradation at perinuclear aggresomes. Disruption of this process leads to dysfunctional endosome accumulation and increased protein aggregation in the cell cytoplasm, both pathological features of neurodegenerative diseases. However, the exact mechanism of dynein functionality in these pathways is still being elucidated. Here, we show that the scaffolding protein SQSTM1 directly interacts with dynein through a previously unidentified dynein-binding site. This interaction is independent of HDAC6, a known interacting protein of both SQSTM1 and dynein. However, knockdown of HDAC6 increases the interaction of SQSTM1 with dynein, indicating a possible competitive interaction. Using different dynein cargoes, we show that SQSTM1 is required for proper dynein motility and trafficking along microtubules. Based on our results,...

Biomedical letters, 1993

... Tyrosine protein kinase activation contributes to ethanol enhanced morphological differentiat... more ... Tyrosine protein kinase activation contributes to ethanol enhanced morphological differentiation of PC12 cells. Auteur(s) / Author(s). WOOTEN MW ; WHITE KR ; LAMAR SEIBENHENER M. ;LLOYD ED ; Affiliation(s) du ou des auteurs / Author(s) Affiliation(s). ...

PLoS ONE, 2013

Protein aggregates can form in the cytoplasm of the cell and are accumulated at aggresomes locali... more Protein aggregates can form in the cytoplasm of the cell and are accumulated at aggresomes localized to the microtubule organizing center (MTOC) where they are subsequently degraded by autophagy. In this process, aggregates are engulfed into autophagosomes which subsequently fuse with lysosomes for protein degradation. A member of the class II histone deacetylase family, histone deacetylase 6(HDAC6) has been shown to be involved in both aggresome formation and the fusion of autophagosomes with lysosomes making it an attractive target to regulate protein aggregation. The scaffolding protein sequestosome 1(SQSTM1)/p62 has also been shown to regulate accumulation and autophagic clearance of protein aggregates. Recent studies have revealed colocalization of HDAC6 and p62 to ubiquitinated mitochondria, as well as, ubiquitinated protein aggregates associated with the E3 ubiquitin ligase TRIM50. HDAC6 deacetylase activity is required for aggresome formation and can be regulated by protein interaction with HDAC6. Due to their colocalization at ubiquitinated protein aggregates, we sought to examine if p62 specifically interacted with HDAC6 and if so, if this interaction had any effect on HDAC6 activity and/or the physiological function of cortactin-F-actin assembly. We succeeded in identifying and mapping the direct interaction between HDAC6 and p62. We further show that this interaction regulates HDAC6 deacetylase activity. Data are presented demonstrating that the absence of p62 results in hyperactivation of HDAC6 and deacetylation of a-tubulin and cortactin. Further, upon induction of protein misfolding we show that p62 is required for perinuclear co-localization of cortactin-F-actin assemblies. Thus, our findings indicate that p62 plays a key role in regulating the recruitment of F-actin network assemblies to the MTOC, a critical cellular function that is required for successful autophagic clearance of protein aggregates.

Molecular and Cellular Biology, 2004

Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays ... more Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.

Molecular and Cellular Biology, 2000

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) si... more The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-ι was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-ι with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-ι by NGF. At the level of Raf-1, neither PKC-ι nor PI3 kinase was required for activation; however, PKC-ι could weakly activate MEK. Inhibitors of PKC-ι activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-ι were likewise required for NGF-induced NF-κB activation...

Molecular and Cellular Biology, 2001

Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated dif... more Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-ι becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-ι were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-ι in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-ι were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-ι. Recruitment of PKC-ι into the complex was dependent on the tyrosine phosphorylation state of PKC-ι. The association of src and PKC-ι was constitutive but was enhanced by NGF treatment, with the src homology 3 domain inter...

Journal of Neuroscience Research, 1997

In an effort to understand the role of protein kinase C (PKC) in nerve growth factor-induced diff... more In an effort to understand the role of protein kinase C (PKC) in nerve growth factor-induced differentiation, we studied the expression of PKC using isoform-specific antibodies. Western blot analysis on whole cell lysates showed that alpha,beta,gamma,epsilon,zeta, iota/lambda and mu were expressed in PC12 cells, except for theta which was absent. In nuclei obtained from control PC12 cells, small amounts of delta, epsilon, iota/lambda and zeta were detected. A computer-assisted search algorithm was used to search for the presence of bipartite nuclear targeting motifs. In classical PKC isoforms alpha,beta,gamma, two bipartite motifs were present, while atypical iota/lambda and zeta-PKC displayed one motif, whereas novel PKC isoforms did not exhibit any bipartite motif structure. Treatment of cells with differentiating doses of nerve growth factor (NGF) resulted in changes of differential magnitude for all of the nuclear PKC isoforms in response to NGF. However, little change in gamma-PKC was observed in response to NGF. This analysis indicated that other factors may contribute to transport of PKC into the nucleus, in addition to the bipartite motif itself. Atypical zeta-PKC is required for NGF-induced neurite outgrowth of PC12 cells (Coleman and Wooten: J Mol Neurosci 5:39-57, 1994). Increases in nuclear zeta-PKC were NGF dose-dependant with a concomitant decrease in cytoplasmic immunoreactivity. The localization of zeta-PKC was investigated by means of immunoelectron microscopy which revealed the localization of this isoform within the inner nuclear matrix bound to chromatin. Taken together, these findings suggest that zeta-PKC may be involved in the regulation of nuclear processes.

Journal of Neuroscience Research, 1999

Both protein kinase C (PKC) and ceramide play a critical role in cell signaling, but the relation... more Both protein kinase C (PKC) and ceramide play a critical role in cell signaling, but the relationship between PKC and ceramide is unclear. Low concentrations of ceramide were observed to transiently stimulate PKC ζ activity in vitro and in vivo, whereas high doses of ceramide lead to inhibition of PKC ζ. Inhibition of activity was accompanied by enhanced binding of the negative regulator, Par4 to PKC ζ. Treatment of PC12 cells with low doses of ceramide promoted survival in serum‐free media and activation of nuclear factor‐κB, whereas higher doses (>2.5 μM) resulted in cell death. Overexpression of either aPKC isoform, PKC ζ or ι, resulted in enhanced survival of PC12 cells at high doses of ceramide and in ceramide‐stimulated Jun N‐terminal kinase (JNK), without any apparent effect on mitogen‐activated kinase. These findings support a role for ceramide‐induced PKC ζ activity in the control of cell survival signaling via a pathway that also activates JNK kinase. J. Neurosci. 55:293–302, 1999. © 1999 Wiley‐Liss, Inc.

Journal of Neurochemistry, 2002

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Journal of Neurochemistry, 2008

Sequestosome 1/p62 was cloned as the interacting partner of the atypical protein kinase C i/k (aP... more Sequestosome 1/p62 was cloned as the interacting partner of the atypical protein kinase C i/k (aPKCs; Sanchez et al. 1998) and has been shown to contain several motifs that enable the protein to serve as a signaling scaffold and as a polyubiquitin binding receptor (Moscat et al. 2007). Recently, we reported that ubiquitinated tau interacts with p62 (Babu et al. 2005). Abnormal phosphorylation of tau is known to promote conformational changes leading to reduced binding to microtubules (Alonso et al. 1994). One hallmark of Alzheimer's disease (AD) is the presence of neurofibrillary tangles (NFTs) containing accumulated hyperphosphorylated polyubiquitinated microtubule associated protein, tau. In brain of individuals with AD, the signaling adapter p62 and the aPKC i/k (Shao et al. 2006) co-localize in neurons with hyperphosphorylated tau that exhibit NFT pathology (Kuusisto et al. 2002). Hyperphosphorylation of tau is one major factor that is believed to drive its aggregation and misfolding (Alonso et al. 1994). The phenotype of mice deficient in the p62 gene is complex reflecting the different critical roles of this protein

Journal of Cellular Biochemistry, 2001

Atypical protein kinase Cs zeta and lambda/iota play a functional role in the regulation of NGF-i... more Atypical protein kinase Cs zeta and lambda/iota play a functional role in the regulation of NGF-induced differentiation and survival of pheochromocytoma, PC12 cells [Coleman and Wooten, 1994; Wooten et al., 1999]. Here we demonstrate an NGF-dependent interaction of aPKC with its binding protein, ZIP/p62. Although, ZIP/p62 was not a PKC-i substrate, the formation of a ZIP/p62-aPKC complex in PC12 cells by NGF occurred post activation of PKC-i and was regulated by the tyrosine phosphorylation state of aPKC. Furthermore, NGF-dependent localization of ZIP/p62 was observed within vesicular structures, identi®ed as late endosomes by colocalization with a Rab7 antibody. Both ZIP/p62 as well as PKC-i colocalized with Rab7 upon NGF stimulation. Inhibition of the tyrosine phosphorylation state of PKC-i did not prevent movement of ZIP/p62 to the endosomal compartment. These observations indicate that the subcellular localization of ZIP/p62 does not depend entirely upon activation of aPKC itself. Of functional importance, transfection of an antisense p62 construct into PC12 cells signi®cantly diminished NGF-induced neurite outgrowth. Collectively, these ®ndings demonstrate that ZIP/p62 acts as a shuttling protein involved in routing activated aPKC to an endosomal compartment and is required for mediating NGF's biological properties.

Journal of Cellular Biochemistry, 2002

Herein, we employed a combined approach of molecular modeling and site-directed mutagenesis to ad... more Herein, we employed a combined approach of molecular modeling and site-directed mutagenesis to address the role of tyrosine phosphorylation in transport of atypical protein kinase C (aPKC) into the nucleus. Computer modeling of the three-dimensional structure of the aPKC catalytic core, reveals that tyrosine 256 (Tyr256) is located at the lip of the activation loop and is conserved among members of the aPKC family, iota/lambda and zeta. Based on these findings, we examined whether tyrosine phosphorylation of aPKC on the activation lip may facilitate nuclear import. An antiserum was generated that selectively recognizes the phosphorylated Tyr256 residue in aPKC. By isolating nuclei of PC12 cells and immunoprecipitating aPKC with Ab-PY256, we observed that Tyr256 is rapidly phosphorylated upon NGF treatment prior to entry of aPKC into the nucleus. aPKC was observed to exclusively bind to importin-beta. The interaction between importin-beta and aPKC was enhanced upon tyrosine phosphorylation of aPKC and binding was abrogated when Tyr256 was mutated to phenylalanine. We propose that phosphorylation of aPKC at Tyr256 induces a conformation, whereby, the arginine-rich NLS is exposed, which then binds importin-beta leading to import of aPKC into the nucleus. Altogether, these findings document a novel role for the tyrosine phosphorylation in regulating import of atypical PKC into the nucleus.

Journal of Biomedicine and Biotechnology, 2006

Aggregated misfolded proteins are hallmarks of most neurodegenerative diseases. In a chronic dise... more Aggregated misfolded proteins are hallmarks of most neurodegenerative diseases. In a chronic disease state, including pathologic situations of oxidative stress, these proteins are sequestered into inclusions. Accumulation of aggregated proteins can be prevented by chaperones, or by targeting their degradation to the UPS. If the accumulation of these proteins exceeds their degradation, they may impair the function of the proteasome. Alternatively, the function of the proteasome may be preserved by directing aggregated proteins to the autophagy-lysosome pathway for degradation. Sequestosome 1/p62 has recently been shown to interact with polyubiquitinated proteins through its UBA domain and may direct proteins to either the UPS or autophagosome. P62 is present in neuronal inclusions of individuals with Alzheimer's disease and other neurodegenerative diseases. Herein, we review p62's role in signaling, aggregation, and inclusion formation, and specifically as a possible contribu...

Journal of Biological Chemistry, 1997

We have previously shown that protein kinase C (PKC)-is activated and required for nerve growth f... more We have previously shown that protein kinase C (PKC)-is activated and required for nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma PC12 cells (

Journal of Biological Chemistry, 2001

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the trans... more Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor B (NF-B). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-B pathway, which mediates NGF survival and differentiation responses. * This study was funded by grants from the National Institutes of Health (to M. W. W.), the Ministry of Science and Education Spain (to M. W. W. and J. M.), and the European Union and Glaxo Wellcome Spain (to J. M.

Journal of Biological Chemistry, 2005