Biomedical subjects

P Garcia-Paramio

Publications and source records attributed to P Garcia-Paramio.

4 recordsLinked to original sources

PubMed1999

Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes.

The generation of antisera specific for the priming phosphorylation sites on protein kinase Calpha (PKCalpha) has permitted analysis of the dephosphorylation of these sites in relation to the down-regulation of the protein. It was demonstrated that these priming sites are subject to agonist-induced dephosphorylation, consistent with inactivation of the protein. Further, the process is shown to be blocked by a PKC inhibitor, indicating a requirement for PKC catalytic activity. This was corroborated by showing that a constitutively active fragment of PKCalpha is able to stimulate the dephosphorylation of wild-type PKCalpha in transfected cells. Consistent with a membrane-traffic event, the process controlled by PKC that leads to dephosphorylation is shown to be temperature-sensitive and to correlate with transient accumulation of PKCalpha on cytoplasmic vesicular structures. It was established that the dephosphorylation of priming sites in PKCalpha is not unique and occurs with other conventional PKC isotypes, demonstrating that this is a general desensitization process for this subclass of kinases. The physiological importance of this desensitization is evidenced by the behaviour of PKCbeta1 in U937 cells, where dephosphorylation of the activation loop site is shown to be a function of cell density.

Amino Acid Sequence↗

PubMed1999

Src promotes PKCdelta degradation.

Platelet-derived growth factor BB (PDGF) stimulates DNA synthesis through a mechanism that is at least partially dependent upon Src family tyrosine kinases, although the signal transduction pathway downstream of Src is poorly understood. We have studied the signaling between Src and different protein kinase C (PKC) isoforms and its possible role in the regulation of PDGF-stimulated DNA synthesis. We found that Src promoted the tyrosine phosphorylation of PKCdelta, and its subsequent degradation. Enforced expression of PKCdelta inhibited PDGF-stimulated DNA synthesis, whereas expression of PKCalpha and PKCepsilon did not, a finding consistent with a model in which PKCdelta negatively regulates G1-to-S-phase progression. We used mutagenesis to map a critical Src phosphorylation site on PKCdelta to tyrosine 311. A mutant form of PKCdelta in which tyrosine 311 was replaced with phenylalanine (Y311F) was more stable in the presence of Src, suggesting that Src-induced degradation was a direct result of PKCdelta tyrosine phosphorylation. We conclude that PKCdelta is downstream of Src but is unlikely to play a positive role in the signaling pathway by which Src promotes DNA synthesis.

3T3 Cells↗

PubMed1998

The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation.

Dominant negative properties are conferred on protein kinase (PK) Calpha by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of such mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-transfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resulted in dominant negative properties. These properties were not restricted to the cognate PKC isotypes, but were effective across the different subclasses. For example, two PKCzeta mutants (atypical isotype) inhibited both PKCalpha (classical isotype) and PKCepsilon (novel isotype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKCalpha, consistent with the expected mode of action. In the case of the PKCalpha mutant, it was shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the phosphorylation of all PKC isotypes.

Animals↗

PubMed1993

Alteration of protein kinase C activity in diabetic rat prostate.

Protein kinase C activity is present in rat prostatic epithelial cells in both cytosolic and membrane subcellular fractions. Partial purification by ion-exchange chromatography and characterization of cofactor requirements showed its behavior as a classical Ca(2+)- and phospholipid-dependent enzyme activated by 1,2-diacylglycerol (or by mimicking agents such as tumor-promoting phorbol esters). Streptozotocin-induced diabetes resulted in an increase of the membrane/cytosolic enzyme ratio suggesting a redistribution of protein kinase C from the cytosolic to the membrane fraction (an index of enzyme activation) that could be reversed toward control conditions by insulin treatment. Differences observed in cofactor requirements for maximal enzyme activation argue for a some distinct expression of protein kinase C isozymes in control and diabetic conditions. These results are a new aspect of the complex set of alterations exhibited by the diabetic prostate in the signal transduction mechanisms that mediate cell functions and proliferation in this gland which could be related to the prostate atrophy and impaired fertility characteristic of this disease.

Animals↗