Centrosomal Anchoring of Protein Kinase C βII by Pericentrin Controls Microtubule Organization, Spindle Function, and Cytokinesis

Chen, Dan; Purohit, Aruna; Halilovic, Ensar; Doxsey, Stephen; Newton, Alexandra C.

doi:10.1074/jbc.m311196200

Cited by 91 publications

(74 citation statements)

References 49 publications

(50 reference statements)

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“…Because expression of the HA-tagged PH domain of PHLPP1 was not detected in cells, the PH domain was expressed as a GST fusion protein for the pull-down experiments. The expression plasmids of various GST-tagged domains of PKC ␤II including N/ (NH 2 terminus plus pseudosubstrate), C1A (C1A domain), C1B (C1B domain), C1AB (C1A plus C1B domains), NC1 (NH 2 terminus plus the entire C1 domain), C2 (C2 domain), KD (kinase domain), and CT (COOH terminus) were constructed as described previously (20,21). The full-length human PP2C␣ cDNA was cloned by one-step reverse transcriptase-PCR using human brain total RNA as template.…”

Section: Methodsmentioning

confidence: 99%

The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

Gao

Brognard

Newton

2008

Journal of Biological Chemistry

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184

198

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The life cycle of protein kinase C (PKC) is controlled by multiple phosphorylation and dephosphorylation steps. The maturation of PKC requires three ordered phosphorylations, one at the activation loop and two at COOH-terminal sites, the turn motif and the hydrophobic motif, to yield a stable and signalingcompetent enzyme. Dephosphorylation of the enzyme leads to protein degradation. We have recently discovered a novel family of protein phosphatases named PH domain leucine-rich repeat protein phosphatase (PHLPP) whose members terminate Akt signaling by dephosphorylating the hydrophobic motif on Akt. Here we show that the two PHLPP isoforms, PHLPP1 and PHLPP2, also dephosphorylate the hydrophobic motif on PKC ␤II, an event that shunts PKC to the detergent-insoluble fraction, effectively terminating its life cycle. Deletion mutagenesis reveals that the PH domain is necessary for the effective dephosphorylation of PKC ␤II by PHLPP in cells, whereas the PDZbinding motif, required for Akt regulation, is dispensable. The phorbol ester-mediated dephosphorylation of the hydrophobic site, but not the turn motif or activation loop, is insensitive to okadaic acid, consistent with PHLPP, a PP2C family member, controlling the hydrophobic site. In addition, knockdown of PHLPP expression reduces the rate of phorbol ester-triggered dephosphorylation of the hydrophobic motif, but not turn motif, of PKC ␣. Last, we show that depletion of PHLPP in colon cancer and normal breast epithelial cells results in an increase in conventional and novel PKC levels. These data reveal that PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation.Protein phosphorylation defines one of the most important and pervasive regulatory mechanisms in cell signaling. Crucial cellular decisions such as those between death or survival and proliferation or differentiation are made depending on the phosphorylation state of signaling molecules. Thus, precise control of the balance between phosphorylation and dephosphorylation is critical for living organisms to maintain normal physiological functions. Dysregulation of signaling pathways that results in disturbing this balance leads to the development of diseases such as cancer and diabetes. Loss of control of either phosphorylation or dephosphorylation mechanisms leads to pathogenic states, and both kinases and phosphatases have been identified as oncogenes or tumor suppressors (1).We have recently identified a novel family of Ser/Thr phosphatases, which we named PH domain leucine-rich repeat protein phosphatase (PHLPP) 3 based on domain composition (2, 3). PHLPP comprises three isozymes: the alternatively spliced PHLPP1␣ and PHLPP1␤, which differ in an amino-terminal extension on PHLPP1␤, and PHLPP2. PHLPP1 and PHLPP2 share 50% overall identity at the amino acid level. PHLPP1 and PHLPP2 contain a PP2C-like phosphatase domain and they dephosphorylate Akt in vitro in a Mn 2ϩ -dependent manner (2, 3). Althoug...

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Section: Methodsmentioning

confidence: 99%

The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

Gao

Brognard

Newton

2008

Journal of Biological Chemistry

Self Cite

184

198

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“…As a result, at least a part of the mutant protein can be 'held' on centrosomes stably. Moreover, many pericentrin-associated proteins, such as dynein and PKC [8,30], predominantly localize in cytoplasm and could retain pericentrin in cytoplasm by interacting with it, although its NES signals were destroyed. Although pericentrin appears predominantly cytoplasmic, it can still shuttle between the cytoplasm and the nucleus.…”

Section: Discussionmentioning

confidence: 99%

“…The NLS-deficient mutant and NESs-deficient mutant of full-length pericentrin were generated by site-directed mutagenesis. The truncated mutants PCNT , PCNT [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] , PCNT [6][7][8][9][10][11][12][13] , PCNT [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] and PCNT [34][35][36][37][38][39][40][41]…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Similar to the yeast homolog, Spc110p, pericentrin anchors γ-tubulin ring complex to centrosomes through interaction with γ-tubulin complex proteins 2 and 3, providing the microtubule nucleation sites and mediating the spindle organization in mitotic cells [5,6]. Several other proteins including cAMPdependent protein kinase (PKA), protein kinase C (PKC) and disrupted-In-schizophrenia 1 (DISC-1) are also recruited to centrosomes by pericentrin [7][8][9]. Moreover, pericentrin could form functional complex with pericentriolar material 1 (PCM1) at centrosomes [10].…”

Section: Introductionmentioning

confidence: 99%

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Pericentrin contains five NESs and an NLS essential for its nucleocytoplasmic trafficking during the cell cycle

Liu

Zhuo

et al. 2010

Cell Res

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Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B (LMB), a specific inhibitor of Crm1, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals (NESs) in pericentrin with diverse export activities. All of the five NESs could bind to Crm1 in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal (NLS) consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.

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“…Conversely, centrosomal abnormalities, early predictors of carcinogenesis, promote mitotic spindle aberrations and chromosome instability, events which are frequently observed in neoplastic cells (27)(28)(29)(30)(31). It is well established that pericentrin supports the normal functioning of the centrosomes and the cytoskeleton and that its function is important to cell cycle progression (27,32,33). Despite the evident functional link of MT1-MMP activity with the cleavage of pericentrin observed in both human and canine cells (18), there were suspicions that MT1-MMP is indirectly, rather than directly, involved in these unorthodox, intracellular, proteolytic events.…”

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confidence: 99%

Centrosomal Pericentrin Is a Direct Cleavage Target of Membrane Type-1 Matrix Metalloproteinase in Humans but Not in Mice

Golubkov

Chekanov

Doxsey

et al. 2005

Journal of Biological Chemistry

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Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits distinctive and important pericellular cleavage functions. Recently, we determined that MT1-MMP was trafficked to the centrosomes in the course of endocytosis. Our data suggested that the functionally important, integral, centrosomal protein, pericentrin-2, was a cleavage target of MT1-MMP in human and in canine cells and that the sequence of the cleavage sites were ALRRLLG 1156 2L 1157 FG and ALRRLLS 2068 2L 2069 FG, respectively. The presence of Asp-948 at the P1 position inactivated the corresponding site (ALRRLLD 948 -L 949 FGD) in murine pericentrin. To confirm that MT1-MMP itself cleaves pericentrin directly, rather than indirectly, we analyzed the cleavage of the peptides that span the MT1-MMP cleavage site. In addition, we analyzed glioma U251 cells, which co-expressed MT1-MMP with the wild type murine pericentrin and the D948G mutant. We determined that the D948G mutant that exhibited the cleavage sequence of human pericentrin was sensitive to MT1-MMP, whereas unmodified murine pericentrin was resistant to proteolysis. Taken together, our results confirm that MT1-MMP cleaves pericentrin-2 in humans but not in mice and that mouse models of cancer probably cannot be used to critically examine MT1-MMP functionality. MT1-MMP2 /MMP-14 is a prototypic member of the membranetethered matrix metalloproteinases (1). Although MT1-MMP is present in normal tissues, its enhanced expression is directly linked to tumor progression and metastasis (2-6). Cell surface-associated MT1-MMP is a multifunctional enzyme (7), and it is involved in the pericellular proteolysis of the extracellular matrix, the activation of soluble MMPs, and the cleavage of adhesion and signaling cell receptors (8 -10). The functional activity of MT1-MMP is regulated by its activation by furinlike proprotein convertases, by its inhibition by TIMPs, and by its selfproteolysis and shedding (11-13). Evidence is also emerging that exocytosis, endocytosis, and recycling also regulate the presentation of MT1-MMP on the cell surface and, consequently, its cell surface-associated proteolytic activity (14 -17).Recently, we determined that functionally active MT1-MMP, which was presented on the cell surface, was internalized, trafficked alongside the microtubular cytoskeleton, and delivered to the centrosomal compartment (16,18). The presence of MT1-MMP in the pericentrosomal space correlated with the cleavage of human pericentrin-2 (kendrin), an integral and functionally important centrosomal, 3336-amino-acid residue long, protein (19 -22), and chromosome instability in non-malignant epithelial Madin-Darby canine kidney cells (18,23). Centrosomes, spindle pole bodies, are cellular organelles that exhibit an ability to organize microtubules and to nucleate (24). The normal functionality of centrosomes is essential to the organization of the cytoskeleton and the mitotic spindle, self-duplication, and cell cycle progression (25, 26). Conversely, centrosomal abnormalities, early predictors of carcinog...

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Centrosomal Anchoring of Protein Kinase C βII by Pericentrin Controls Microtubule Organization, Spindle Function, and Cytokinesis

Cited by 91 publications

References 49 publications

The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

Pericentrin contains five NESs and an NLS essential for its nucleocytoplasmic trafficking during the cell cycle

Centrosomal Pericentrin Is a Direct Cleavage Target of Membrane Type-1 Matrix Metalloproteinase in Humans but Not in Mice

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