Flowchart: Preparation: Camk2

 

Calcium/Calmodulin and Calmodulin Kinase II Stimulate Hyperactivation in Demembranated Bovine Sperm.

Ignotz GG, Suarez SS.

Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853.

Hyperactivated motility is observed among sperm in the mammalian oviduct near the time of ovulation. It is characterized by high-amplitude, asymmetrical flagellar beating and assists sperm in penetrating the cumulus oophorus and zona pellucida. Elevated intracellular Ca(2+) is required for the initiation of hyperactivated motility, suggesting that calmodulin (CALM) and Ca(2+)/CALM-stimulated pathways are involved. A demembranated sperm model was used to investigate the role of CALM in promoting hyperactivation. Ejaculated bovine sperm were demembranated and immobilized by brief exposure to Triton X-100. Motility was restored by addition of reactivation medium containing MgATP and Ca(2+), and hyperactivation was observed as free Ca(2+) was increased from 50 nM to 1 muM. However, when 2.5 mM Ca(2+) was added to the demembranation medium to extract flagellar CALM, motility was not reactivated unless exogenous CALM was readded. The inclusion of anti-CALM IgG in the reactivation medium reduced the proportion hyperactivated in 1 muM Ca(2+) to 5%. Neither control IgG, the CALM antagonist W-7, nor a peptide directed against the CALM-binding domain of myosin light chain kinase (MYLK2) inhibited hyperactivation. However, when sperm were reactivated in the presence of CALM kinase II (CAMK2) inhibiting peptides, hyperactivation was reduced by 75%. Furthermore, an inhibitor of CAMK2, KN-93, inhibited hyperactivation without impairing normal motility of intact sperm. CALM and CAMK2 were immunolocalized to the acrosomal region and flagellum. These results indicate that hyperactivation is stimulated by a Ca(2+)/CALM pathway involving CAMK2.

PMID: 15878888 [PubMed - in process

 

The polo-like kinase Plx1 prevents premature inactivation of the APC(Fizzy)-dependent pathway in the early Xenopus cell cycle.

Brassac T, Castro A, Lorca T, Le Peuch C, Doree M, Labbe JC, Galas S.

Centre de Recherches de Biochimie Macromoleculaire, CNRS UPR 1086, Montpellier, France.

Members of the polo-like family of protein kinases have been involved in the control of APC (anaphase-promoting complex) during the cell cycle, yet how they activate APC is not understood in any detail. In Xenopus oocytes, Ca2+-dependent degradation of cyclin B associated with release from arrest at second meiotic metaphase was demonstrated to require the polo-like kinase Plx1. The aim of the present study was to examine, beyond Ca2+-dependent resumption of meiosis, the possible role of Plx1 in the control of cyclin degradation during the early mitotic cell cycle. Plx1 was found to be dispensable for MPF to turn on the cyclin degradation machinery. However, it is required to prevent premature inactivation of the APC-dependent proteolytic pathway. Microcystin suppresses the requirement for Plx1 in both Ca2+-dependent exit from meiosis, associated with degradation of both cyclin B and A downstream of CaMK2 activation, and prevention of premature APC(Fizzy) inactivation in the early mitotic cell cycle. These results are consistent with the view that Plx1 antagonizes an unidentified microcystin-sensitive phosphatase that inactivates APC(Fizzy).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IGF-1 protects cardiac myocytes from hyperosmotic stress-induced apoptosis via CREB.

Maldonado C, Cea P, Adasme T, Collao A, Diaz-Araya G, Chiong M, Lavandero S.

Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Quimicas y Farmaceuticas, Universidad de Chile, Santiago, Chile; Centro FONDAP Estudios Moleculares de la Celula, Universidad de Chile, Santiago, Chile.

Hyperosmotic stress stimulates a rapid and pronounced apoptosis in cardiac myocytes which is attenuated by insulin-like growth factor-1 (IGF-1). Because in these cells IGF-1 induces intracellular Ca(2+) increase, we assessed whether the cyclic AMP response element-binding protein (CREB) is activated by IGF-1 through Ca(2+)-dependent signalling pathways. In cultured cardiac myocytes, IGF-1 induced phosphorylation (6.5+/-1.0-fold at 5min), nuclear translocation (30min post-stimulus) and DNA binding activity of CREB. IGF-1-induced CREB phosphorylation was mediated by MEK1/ERK, PI3-K, p38-MAPK, as well as Ca(2+)/calmodulin kinase and calcineurin. Exposure of cardiac myocytes to hyperosmotic stress (sorbitol 600mOsm) decreased IGF-1-induced CREB activation Moreover, overexpression of a dominant negative CREB abolished the anti-apoptotic effects of IGF-1. Our results suggest that IGF-1 activates CREB through a complex signalling pathway, and this transcription factor plays an important role in the anti-apoptotic action of IGF-1 in cultured cardiac myocytes.

PMID: 16168389 [PubMed - as supplied by publisher]

 

 

 

 

 

 
 


Text Box: Plx1                                                    

Text Box: Apc                  

                            

                       

Text Box: CamkII                                                               

 

                                                             

                                                                    

 

Text Box: CrebText Box: Cycline BText Box: Cycline A