Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization

2001 ◽  
Vol 114 (9) ◽  
pp. 1787-1794 ◽  
Author(s):  
G.Z. Zhu ◽  
D.G. Myles ◽  
P. Primakoff
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Acrosome Reaction ◽  
Phosphorylation Site ◽  
Equatorial Region ◽  
Proteolytic Processing ◽  
Mature Sperm ◽  
Kinase C

Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin (α) and (β), cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs' protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.

scholarly journals Phosphorylation of GAP-43 (growth-associated protein of 43 kDa) by conventional, novel and atypical isotypes of the protein kinase C gene family: differences between oligopeptide and polypeptide phosphorylation

1996 ◽  
Vol 317 (1) ◽  
pp. 219-224 ◽  
Author(s):  
Silke A. OEHRLEIN ◽  
Peter J. PARKER ◽  
Thomas HERGET
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mammalian Brain ◽  
Major Protein ◽  
Calmodulin Binding ◽  
Kinase C ◽  
Significant Difference ◽  
Complete Protein

GAP-43 (growth-associated protein of 43 kDa; also known as neuromodulin, P-57, B-50 and F-1) is a neuronal calmodulin binding protein and a major protein kinase C (PKC) substrate in mammalian brain. Here we describe the phosphorylation by and the site specificity of different PKC isotypes. The conventional PKC β1 and the novel PKCs Δ and ϵ effectively phosphorylated recombinant GAP-43 in vitro; atypical PKC ζ did not. The Km values (between 0.6 and 2.3 μM) were very low, demonstrating a high-affinity interaction between kinase and substrate. All PKC isotypes were shown to phosphorylate serine-41 in GAP-43. When using a 19-amino-acid oligopeptide based on the GAP-43 phosphorylation site as substrate, there was a significant difference compared with polypeptide phosphorylation. The Vmax values of PKC β1 and PKC ϵ were much higher for this oligopeptide than for the complete protein (up to 10-fold); in contrast, their apparent affinities for the peptide were much lower (up to 100-fold) than for the intact GAP-43 polypeptide. Furthermore, phosphorylation of the GAP-43 oligopeptide by PKC β1 was more sensitive to a catalytic-site inhibitor than was phosphorylation of intact GAP-43. These results suggest that there are multiple sites of interaction between GAP-43 and PKC.

scholarly journals Inhibition of insulin-stimulated phosphorylation of the intracellular domain of phospholemman decreases insulin-dependent GLUT4 translocation in streptolysin-O-permeabilized adipocytes

1999 ◽  
Vol 343 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Otto WALAAS ◽  
Robert S. HORN ◽  
S. Ivar WALAAS
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Glucose Transporter ◽  
Insulin Signalling ◽  
Glut4 Translocation ◽  
Intracellular Domain ◽  
Kinase C ◽  
Streptolysin O

A variety of studies indicate that protein kinase C might be involved in the insulin signalling cascade leading to translocation of the insulin-regulated glucose transporter GLUT4 from intracellular pools to the plasma membrane. Phospholemman is a plasma-membrane protein kinase C substrate whose phosphorylation is increased by insulin in intact muscle [Walaas, Czernik, Olstad, Sletten and Walaas (1994) Biochem. J. 304, 635-640]. The present study examined whether the inhibition of phospholemman phosphorylation modulates the effects of insulin on GLUT4 translocation. For this purpose, a synthetic peptide derived from the intracellular domain of phospholemman with the phosphorylatable serine residues replaced with alanine residues was prepared. This peptide was found to decrease the protein kinase C-catalysed phosphorylation of a synthetic phospholemman peptide in vitro. When introduced into streptolysin-O-permeabilized adipocytes, the peptide decreased the effects of insulin on both the phosphorylation of phospholemman and the recruitment of GLUT4 to the plasma membrane. Similarly, the internalization of phospholemman antibodies, which also decreased the protein kinase C-mediated phosphorylation of the synthetic phospholemman peptide in vitro, decreased the effect of insulin on GLUT4 translocation in the adipocytes. The results suggest that phosphorylation of the intracellular domain of phospholemman might be involved in modulating the insulin-induced translocation of GLUT4 to the plasma membrane.

scholarly journals Dynamics of Protein Kinase C-mediated Phosphorylation of the Complement C5a Receptor on Serine 334

2006 ◽  
Vol 282 (7) ◽  
pp. 4345-4353 ◽  
Author(s):  
Beatrix Pollok-Kopp ◽  
Friederike Hüttenrauch ◽  
Stephanie Rethorn ◽  
Martin Oppermann
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Phosphorylation Sites ◽  
Recombinant Enzymes ◽  
C5a Receptor ◽  
Kinase C ◽  
Complement C5a ◽  
C5a Anaphylatoxin

Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t½ ∼ 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-β and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nm) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t½ < 5 s) binding of β-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.

In vitro and in vivo suppression of growth of rat liver epithelial tumor cells by antisense oligonucleotide against protein kinase C-alpha

2000 ◽  
Vol 33 (4) ◽  
pp. 601-608 ◽  
Author(s):  
Shwu-Bin Lin ◽  
Li-Ching Wu ◽  
Siao-Ling Huang ◽  
Hui-Lun Hsu ◽  
Sung-Hwa Hsieh ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tumor Cells ◽  
Rat Liver ◽  
Antisense Oligonucleotide ◽  
Epithelial Tumor ◽  
Kinase C ◽  
Epithelial Tumor Cells
Sign in / Sign up

Export Citation Format

Share Document