Heidi Outzen - Academia.edu (original) (raw)

Papers by Heidi Outzen

utophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it ... more utophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/ SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipiA tated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutan...

Background / Purpose: The protein kinase C (PKC) family of serine/threonine kinases consists of t... more Background / Purpose: The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms that can be grouped into three classes denoted classical-, novel- and atypical PKCs. λ/ι and ζPKCs constitute the atypical PKCs, which serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to find novel interaction partners for aPKCs, a yeast two-hybrid screening of a HeLa cell cDNA library was carried out using the regulatory domain of λ/ιPKC as bait. This screen yielded three independent clones of transforming growth factor β inducible early response gene 1 (TIEG1) as a putative interaction partner for λ/ιPKC. Main conclusion: We have confirmed the interaction in vitro and in vivo using pull-down- and co-immunoprecipitation assays and confocal microscopy. In addition, we report that aPKCs phosphorylate the DNA binding domain (DBD) of TIEG1 on t...

Journal of Biological Chemistry, 2007

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubi... more Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related ␥-aminobutyrate receptor-associated protein and ␥-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62-and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62-and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 m diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.

Journal of Biological Chemistry, 2003

The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction dom... more The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, /and PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aP-KCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1996

Four differently charged trypsins were purified from pyloric caeca of Atlantic sahnon (Salmo salar).

Cellular and Molecular Life Sciences, 2011

The protein kinase C (PKC) family of serine/ threonine kinases consists of ten different isoforms... more The protein kinase C (PKC) family of serine/ threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCi/k and PKCf serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCi/k as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCi/k. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential.

Biochemical Journal, 2008

ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK... more ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser186 of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser186, indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser186. This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser186 is replaced with either an alanine residue o...

The Journal of Cell Biology, 2005

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it... more Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant ...

utophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it ... more utophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/ SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipiA tated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutan...

Background / Purpose: The protein kinase C (PKC) family of serine/threonine kinases consists of t... more Background / Purpose: The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms that can be grouped into three classes denoted classical-, novel- and atypical PKCs. λ/ι and ζPKCs constitute the atypical PKCs, which serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to find novel interaction partners for aPKCs, a yeast two-hybrid screening of a HeLa cell cDNA library was carried out using the regulatory domain of λ/ιPKC as bait. This screen yielded three independent clones of transforming growth factor β inducible early response gene 1 (TIEG1) as a putative interaction partner for λ/ιPKC. Main conclusion: We have confirmed the interaction in vitro and in vivo using pull-down- and co-immunoprecipitation assays and confocal microscopy. In addition, we report that aPKCs phosphorylate the DNA binding domain (DBD) of TIEG1 on t...

Journal of Biological Chemistry, 2007

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubi... more Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related ␥-aminobutyrate receptor-associated protein and ␥-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62-and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62-and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 m diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.

Journal of Biological Chemistry, 2003

The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction dom... more The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, /and PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aP-KCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1996

Four differently charged trypsins were purified from pyloric caeca of Atlantic sahnon (Salmo salar).

Cellular and Molecular Life Sciences, 2011

The protein kinase C (PKC) family of serine/ threonine kinases consists of ten different isoforms... more The protein kinase C (PKC) family of serine/ threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCi/k and PKCf serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCi/k as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCi/k. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential.

Biochemical Journal, 2008

ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK... more ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser186 of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser186, indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser186. This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser186 is replaced with either an alanine residue o...

The Journal of Cell Biology, 2005

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it... more Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant ...