Significance of thymosin β4 and implication of PINCH-1-ILK-α-parvin (PIP) complex in human dilated cardiomyopathy

Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression--the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI.     

α1A-Adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α₁-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α₁-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α_1A-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α₁-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α_1A-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α_1A-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α_1A-AR agonism.     

Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway

Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia-induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up-regulation of autophagy-related gene (Atg) expression. Moreover, suppression of autophagy with DN-Atg5(K130R) or 3-methyladenine (3-MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia-induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up-regulated expression of hypoxia-inducible factor-1α (HIF-1α)-dependent Bcl-2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF-1α or BNIP3 obviously abrogated hypoxia-induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia-induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis.     

H89 sensitive kinase regulates the translocation of Sar1 onto the ER membrane through phosphorylation of ER-coupled β-tubulin

ER-to-Golgi protein transport is carried out by transport vesicles which are formed at the ER-exit sites with recruitment of cytoplasmic coat proteins. Vesicle formation is initiated by assembly of the small G protein (Sar1) onto the ER membrane. Sar1 assembly onto the ER membrane is suppressed by protein kinase inhibitor H89, suggesting participation of H89-sensitive kinase in this process. The present study identified an effector of H89-sensitive kinase by LC-MS PMF analysis combined with 1D- and 2D-PAGE autoradiography, and examined the changes on the effector and Sar1 translocation induced by H89. H89 significantly suppressed the phosphorylation of 55 kDa protein with dosage dependency, and phosphorylation of 55 kDa, pI 5.5 protein spot in 2-D-autoradiography was drastically diminished by H89. LC-MS PMF analysis showed that the protein spot was β-tubulin. H89 significantly suppressed Sar1 translocation onto the ER. These findings indicate that β-tubulin is one of downstream effectors of H89-sensitive kinase, and that suppression of ER-coupled β-tubulin phosphorylation decreases Sar1 translocation onto the ER, suggesting that phosphorylation of β-tubulin regulates Sar1 translocation.     

Abl regulates planar polarized junctional dynamics through β-catenin tyrosine phosphorylation

Interactions between epithelial cells are mediated by adherens junctions that are dynamically regulated during development. Here we show that the turnover of β-catenin is increased at cell interfaces that are targeted for disassembly during Drosophila axis elongation. The Abl tyrosine kinase is concentrated at specific planar junctions and is necessary for polarized β-catenin localization and dynamics. abl mutant embryos have decreased β-catenin turnover at shrinking edges, and these defects are accompanied by a reduction in multicellular rosette formation and axis elongation. Abl promotes β-catenin phosphorylation on the conserved tyrosine 667 and expression of an unphosphorylatable β-catenin mutant recapitulates the defects of abl mutants. Notably, a phosphomimetic β-catenin(Y667E) mutation is sufficient to increase β-catenin turnover and rescue axis elongation in abl deficient embryos. These results demonstrate that the asymmetrically localized Abl tyrosine kinase directs planar polarized junctional remodeling during Drosophila axis elongation through the tyrosine phosphorylation of β-catenin.     

α(1A)-adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α?-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α?-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α(1A)-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α?-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α(1A)-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α(1A)-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α(1A)-AR agonism.     

Mitochondrial Lon regulates apoptosis through the association with Hsp60–mtHsp70 complex

Human Lon protease is a mitochondrial matrix protein with several functions, including protein degradation, mitochondrial DNA (mtDNA) binding, and chaperone activity. Lon is currently emerging as an important regulator of mitochondria-contributed tumorigenesis due to its overexpression in cancer cells. To understand the mechanism of increased Lon in tumor cells, we studied the interactome to identify the chaperone Lon-associated proteins by proteomics approaches using the cells overexpressing Lon. In the present study, we designed a method connecting co-immunoprecipitation (Co-IP) to in-solution digestion for the shotgun mass spectrometry. We identified 76 proteins that were putative Lon-associated proteins that participated in mitochondrial chaperone system, cellular metabolism and energy, cell death and survival, and mtDNA stability. The association between Lon and NDUFS8 or Hsp60–mtHsp70 complex was confirmed by Co-IP and immunofluorescence co-localization assay. We then found that the protein stability/level of Hsp60–mtHsp70 complex depends on the level of Lon under oxidative stress. Most importantly, the ability of increased Lon-inhibited apoptosis is dependent on Hsp60 that binds p53 to inhibit apoptosis. These results suggest that the mechanism underlying cell survival regulated by Lon is mediated by the maintenance of the protein stability of Hsp60–mtHsp70 complex. This new knowledge of chaperone Lon interactome will allow us to better understand the cellular mechanism of Lon in mitochondrial function and of its overexpression in enhancing cell survival and tumorigenesis.Under stress circumstances, proteins are at risk of being inactivated by misfolding, unfolding, or aggregation. Protein quality control (PQC) system, chaperones and proteases, safeguards the function under cellular stress conditions. The coordinated function of the two components is required to stabilize misfolded proteins and refold or remove them to avoid the deleterious effects of protein aggregation.^{1, 2}Lon is a highly conserved AAA+ (ATPases associated with a variety of cellular activities) protease and is committed to several crucial functions, including adenosine-5′-triphosphate (ATP)-dependent proteolytic, DNA binding, and chaperone-like activity.^{3, 4, 5} Eukaryotic Lon protease operates in PQC in mitochondria by its multiple functions^{4, 6, 7} and has a critical role in the maintenance of mitochondrial function, biogenesis, and homeostasis.⁸ Mitochondria orchestrate the process of cell life and death, thereby employing a decisive control over signaling leading to cellular survival, in particular the intrinsic pathway of apoptosis.⁹ Thus it is not surprising that the level of Lon regulates mitochondrial functions that contribute to cell fate and survival. Indeed, Lon downregulation leads to loss of mitochondrial function, early embryonic lethality, reduced cell proliferation, and apoptosis.^{10, 11, 12, 13} Lon upregulation is critical for cancer cell survival and tumorigenesis by regulating stress responses induced by oxidative condition.^{11, 12} Lon is a stress protein and induced by a number of stresses such as hypoxia and oxidative and mitochondrial unfolded protein stress,^{11, 14, 15, 16, 17} which are common stress phenotypes of cancer cells. During hypoxia, Lon is upregulated by the hypoxia-inducible factor-1α and is involved in a mechanism to respond to low oxygen availability and adapt cancer cells to a hypoxic environment.¹⁶ In addition to its proteolytic activity, Lon has been found to show chaperone properties.^{3, 11, 15} Lon promotes the assembly of cytochrome c oxidase (COX) 4–1 subunits, suggesting that Lon has chaperone activity in yeast and mammalian cells.^{15, 18} Molecular chaperones of heat-shock protein (HSP) family have important roles in promoting tumor growth and survival.^{19, 20} Thus mitochondrial Lon may be a protein chaperone to assist cells to survive and adapt to various stresses that are linked to oncogenesis. However, very few human Lon chaperone clients have been identified, and the mechanism of how upregulated Lon employs its chaperone activity to regulate apoptosis remains obscure.To study the roles of Lon overexpression in cancer cell survival, we utilized proteomic techniques to identify chaperone Lon-interacting proteins. The interactome suggests that Lon may participate in many cellular activities, including mitochondrial chaperones, cellular metabolism and energy, Redox regulation, cell death and survival, and mitochondrial DNA (mtDNA) stability. We identified heat-shock protein 60 (Hsp60), mtHsp70, and NDUFS8 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 8) as Lon-interacting proteins by using co-immunoprecipitation (Co-IP) and immunofluorescence experiments. We further characterized that the protein stability of Hsp60 and mtHsp70 depends on the level of Lon under oxidative stress. We know the fact that Hsp60 and mtHsp70 forms a complex^{21, 22} and are overexpressed in cancer cells and have crucial roles in modulating the apoptotic pathways and in cancer development.¹⁹ Consistently, Hsp60 is essential to maintain apoptosis inhibition preserved by Lon overexpression. These results suggest that the mechanism underlying apoptosis regulated by Lon is mediated by the maintenance of the stability of Hsp60–mtHsp70 complex.     

αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.     

IL-32θ negatively regulates IL-1β production through its interaction with PKCδ and the inhibition of PU.1 phosphorylation

It has been well known that IL-32 exerts pro-inflammatory effects on the various inflammatory diseases in clinical studies. Here, we confirmed that IL-32θ, a new isoform of IL-32, decreased the phorbol 12-myristate 13-acetate (PMA)-induced IL-1β expression in THP-1 human myelomonocyte. We previously reported that the IL-32 isoforms control expressions of other cytokines via novel PKCs. Likewise, IL-32θ interacted with PKCδ, and consequently inhibited PKCδ-mediated phosphorylation of PU.1. Moreover, IL-32θ attenuated the localization of PU.1 into the IL-1β promoter region. These findings reveal that IL-32θ reduces PKCδ-mediated phosphorylation of PU.1, resulting in attenuation of IL-1β production.     

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin α2

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin α2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin α2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin α2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin α2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin α2β1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.     

Mink1 regulates β-catenin-independent Wnt signaling via Prickle phosphorylation

β-Catenin-independent Wnt signaling pathways have been implicated in the regulation of planar cell polarity (PCP) and convergent extension (CE) cell movements. Prickle, one of the core proteins of these pathways, is known to asymmetrically localize proximally at the adherens junction of Drosophila melanogaster wing cells and to locally accumulate within plasma membrane subdomains in cells undergoing CE movements during vertebrate development. Using mass spectrometry, we have identified the Ste20 kinase Mink1 as a Prickle-associated protein and found that they genetically interact during the establishment of PCP in the Drosophila eye and CE in Xenopus laevis embryos. We show that Mink1 phosphorylates Prickle on a conserved threonine residue and regulates its Rab5-dependent endosomal trafficking, a process required for the localized plasma membrane accumulation and function of Prickle. Mink1 also was found to be important for the clustering of Vangl within plasma membrane puncta. Our results provide a link between Mink and the Vangl-Prickle complex and highlight the importance of Prickle phosphorylation and endosomal trafficking for its function during Wnt-PCP signaling.     

An osteoclastic protein-tyrosine phosphatase regulates the β3-integrin, syk, and shp1 signaling through respective src-dependent phosphorylation in osteoclasts

This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-β(3)-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-β(3) integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-β(3)-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-β(3)-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCγ levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, PTP-oc plays a central regulatory role in the concerted regulation of the β(3)-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through activation of Src PTK.     

VEGF₁₆₄ differentially regulates neutrophil and T cell adhesion through ItgaL- and ItgaM-dependent mechanisms

Leukocyte recruitment to inflamed tissues is the cornerstone of inflammatory responses and the driving force behind the establishment of inflammatory bowel disease, consisting of Crohn's disease and ulcerative colitis. It has been reported that angiogenic cytokines contribute to this inflammatory response that facilitates the chronic nature of disease. We have previously reported (Goebel S, Huang M, Davis WC, Jennings M, Siahaan TJ, Alexander JS, Kevil CG. Am J Physiol Gastrointest Liver Physiol 290: G648-G654, 2006) that vascular endothelial growth factor (VEGF)-A can stimulate neutrophil adhesion to colon microvascular endothelial cells in a β?-integrin (Itgb2)-dependent manner. However, it is not known which of the specific leukocyte integrins are critical for VEGF-A-dependent neutrophil and T cell recruitment. Here we examine the differential importance of either α-integrin (Itga)L or ItgaM in governing neutrophil and T cell adhesion to VEGF-A-activated colonic endothelium. Using an in vitro parallel-plate flow chamber model, we found that genetic deficiency of ItgaM completely blunted neutrophil adhesion to VEGF-A-stimulated endothelium, whereas ItgaL deficiency only partly blocked neutrophil adhesion. Deficiency of ItgaM did significantly decrease neutrophil rolling, whereas deficiency of ItgaL did not. We found that genetic deficiency of either ItgaL or ItgaM did significantly blunt T cell adhesion to VEGF-A-stimulated colon endothelium. We also found that genetic deficiency of these Itgas significantly attenuated T cell rolling behavior. Lastly, we examined whether VEGF-A-mediated leukocyte recruitment occurred through different VEGF receptor (VEGFR) pathways and found that VEGFR2 activation regulates neutrophil recruitment, whereas both VEGFR1 and VEGFR2 modulate T cell recruitment. Together, these data identify differential molecular mechanisms of VEGF-A-mediated leukocyte recruitment.     

Cdk-mediated phosphorylation of the Kvβ2 auxiliary subunit regulates Kv1 channel axonal targeting

Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvβ2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)-mediated phosphorylation at these sites negatively regulates the interaction of Kvβ2 with the microtubule plus end-tracking protein EB1. Endogenous Cdks, EB1, and Kvβ2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvβ2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvβ2 to EB1.     

Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H₂O₂) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H₂O₂ compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.