PTEN-PDZ domain interactions: binding of PTEN to PDZ domains of PTPN13.

Protein modular interactions mediated by PDZ domains are essential for the establishment of functional protein networks controlling diverse cellular functions. The tumor suppressor PTEN possesses a C-terminal PDZ-binding motif (PDZ-BM) that is recognized by a specific set of PDZ domains from scaffolding and regulatory proteins. Here, we review the current knowledge on PTEN-PDZ domain interactions and tumor suppressor networks, describe methodology suitable to analyze these interactions, and report the binding of PTEN and the PDZ domain-containing protein tyrosine phosphatase PTPN13. Yeast two-hybrid and GST pull-down analyses showed that PTEN binds to PDZ2/PTPN13 domain in a manner that depends on the specific PTPN13 PDZ domain arrangement involving the interdomain region between PDZ1 and PDZ2. Furthermore, a specific binding profile of PTEN to PDZ2/PTPN13 domain was observed by mutational analysis of the PTEN PDZ-BM. Our results disclose a PDZ-mediated physical interaction of PTEN and PTPN13 with potential relevance in tumor suppression and cell homeostasis.

A functional network of the tumor suppressors APC, hDlg, and PTEN, that relies on recognition of specific PDZ-domains.

APC and PTEN are tumor suppressor proteins that bind through their C-termini to the PDZ domain containing-hDlg scaffolding protein. We have found that co-expression of PTEN and hDlg enhanced the negative regulation of the PI3K/Akt pathway by PTEN, indicating the physiologic importance of these interactions. APC and PTEN share other PDZ domain containing-interacting partners, including the MAGI scaffolding proteins and the MAST family of protein kinases. Mutational analysis revealed that the C-terminal PDZ-binding motifs from APC and PTEN were differentially recognized by distinct PDZ domains. APC bound to the three PDZ domains from hDlg, whereas PTEN mainly bound to PDZ-2/hDlg. This indicates the existence of overlapping, but distinct PDZ-domain recognition patterns by APC and PTEN. Furthermore, a ternary complex formed by APC, PTEN, and hDlg was detected, suggesting that hDlg may serve as a platform to bring in proximity APC and PTEN tumor suppressor activities. In line with this, tumor-related mutations targeting the PDZ-2/hDlg domain diminished its interaction with APC and PTEN. Our results expand the PDZ-domain counterparts for the tumor suppressor APC, show that APC and PTEN share PDZ-domain partners but have individual molecular determinants for specific recognition of PDZ domains, and suggest the participation of the tumor suppressors APC, PTEN, and hDlg in PDZ-domain interaction networks which may be relevant in oncogenesis.

Nuclear localization of PTEN by a Ran-dependent mechanism enhances apoptosis: Involvement of an N-terminal nuclear localization domain and multiple nuclear exclusion motifs.

The targeting of the tumor suppressor PTEN protein to distinct subcellular compartments is a major regulatory mechanism of PTEN function, by controlling its access to substrates and effector proteins. Here, we investigated the molecular basis and functional consequences of PTEN nuclear/cytoplasmic distribution. PTEN accumulated in the nucleus of cells treated with apoptotic stimuli. Nuclear accumulation of PTEN was enhanced by mutations targeting motifs in distinct PTEN domains, and it was dependent on an N-terminal nuclear localization domain. Coexpression of a dominant negative Ran GTPase protein blocked PTEN accumulation in the nucleus, which was also affected by coexpression of importin alpha proteins. The lipid- and protein-phosphatase activity of PTEN differentially modulated PTEN nuclear accumulation. Furthermore, catalytically active nuclear PTEN enhanced cell apoptotic responses. Our findings indicate that multiple nuclear exclusion motifs and a nuclear localization domain control PTEN nuclear localization by a Ran-dependent mechanism and suggest a proapoptotic role for PTEN in the cell nucleus.

Functional definition of relevant epitopes on the tumor suppressor PTEN protein.

The binding of PTEN to PDZ-domain-containing proteins appears to play an important role in the control of cell growth, motility and apoptosis. In turn, this binding can be abrogated by cleavage of the PTEN C-terminal region by caspase-3. We have generated and characterized monoclonal antibodies (mAb) directed against distinct epitopes at the C-terminal region of PTEN, and used them to define protein-binding epitopes on PTEN and to study its cleavage by caspase-3. mAb directed against epitopes at the far C-terminus of PTEN blocked binding to PTEN cognate PDZ domains and did not recognize the caspase-3 cleaved PTEN fragments. On the other hand, mAb that recognized an epitope within the C2 domain of PTEN did not prevent binding to PDZ domains, but could detect the caspase-3 cleaved PTEN fragments. The analysis of PTEN cleavage by caspase-3 revealed that the lipid phosphatase activity of PTEN controls its own degradation by interfering with the PI3-K anti-apoptotic activity. Our results define protein-binding sites on the PTEN tumor suppressor at the immunochemical level, and suggest a regulatory link between PTEN phosphatase activity, caspase-3 sensitivity and PTEN-protein interactions.

Binding of PTEN to specific PDZ domains contributes to PTEN protein stability and phosphorylation by microtubule-associated serine/threonine kinases.

The tumor suppressor phosphatase PTEN is a key regulator of cell growth and apoptosis that interacts with PDZ domains from regulatory proteins, including MAGI-1/2/3, hDlg, and MAST205. Here we identified novel PTEN-binding PDZ domains within the MAST205-related proteins, syntrophin-associated serine/threonine kinase and MAST3, characterized the regions of PTEN involved in its interaction with distinctive PDZ domains, and analyzed the functional consequences on PTEN of PDZ domain binding. Using a panel of PTEN mutations, as well as PTEN chimeras containing distinct domains of the related protein TPTE, we found that the PTP and C2 domains of PTEN do not affect PDZ domain binding and that the C-terminal tail of PTEN (residues 350-403) provides selectivity to recognize specific PDZ domains from MAGI-2, hDlg, and MAST205. Binding of PTEN to the PDZ-2 domain from MAGI-2 increased PTEN protein stability. Furthermore, binding of PTEN to the PDZ domains from microtubule-associated serine/threonine kinases facilitated PTEN phosphorylation at its C terminus by these kinases. Our results suggest an important role for the C-terminal region of PTEN in the selective association with scaffolding and/or regulatory molecules and provide evidence that PDZ domain binding stabilizes PTEN and targets this tumor suppressor for phosphorylation by microtubule-associated serine/threonine kinases.

8-NH2-boldine, an antagonist of alpha1A and alpha1B adrenoceptors without affinity for the alpha1D subtype: structural requirements for aporphines at alpha1-adrenoceptor subtypes.

Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.

Phosphorylation-regulated cleavage of the tumor suppressor PTEN by caspase-3: implications for the control of protein stability and PTEN-protein interactions.

PTEN phosphatase is one of the most commonly targeted tumor suppressors in human cancers and a key regulator of cell growth and apoptosis. We have found that PTEN is cleaved by caspase-3 at several target sites, located in unstructured regions within the C terminus of the molecule. Cleavage of PTEN was increased upon TNFalpha-cell treatment and was negatively regulated by phosphorylation of the C-terminal tail of PTEN by the protein kinase CK2. The proteolytic PTEN fragments displayed reduced protein stability, and their capability to interact with the PTEN interacting scaffolding protein S-SCAM/MAGI-2 was lost. Interestingly, S-SCAM/MAGI-2 was also cleaved by caspase-3. Our findings suggest the existence of a regulatory mechanism of protein stability and PTEN-protein interactions during apoptosis, executed by caspase-3 in a PTEN phosphorylation-regulated manner.

Asma y Herencia / Asthma and inheritance

Se plantea que el papel de la herencia en la etiología del asma bronquial ha sido objeto de estudio desde hace muchos años. Se realiza esta investigación en niños cubanos con antecedentes familiares de alergia y se analizan la edad en que comenzaron a presentar síntomas de asma bronquial, así como la herencia y otros factores desfavorables que ejercen influencia en el desarrollo de esta enfermedad

Rehabilitación del paciente asmático con deformidad torácica / Rehabilitation of the asthmatic with thoracic deformity

Se realizó un estudio en pacientes asmáticos grado III que presentaban deformidades torácicas como complicación, y en el cual se les indicaron pruebas funcionales respiratorias, medidas antropométricas y resistencia pulmonar antes de un tratamiento fisioterapéutico y 2 años después, dirigido a fortalecer la musculatura respiratoria, el ritmo respiratorio y corregir las deformidades torácicas y posturales. Se expresa que transcurridos 2 años se encontró que, en el 90

de los pacientes, estas deformidades habían mejorado significativamente; se señala que estos niños tenían como único tratamiento medicamentoso el intal. Se apreció, en el 100

de los infantes, una mejoría clínica evidente de sus síntomas respiratorios, una disminución significativa de la resistencia de las vías aéreas y un aumento marcado de la capacidad vital. Se concluye que la fisioterapia y los ejercicios respiratorios son de utilidad en el tratamiento a pacientes asmáticos con deformidades torácicas (AU)

Reactividad cutánea a antígenos bacterianos durante el primer año de vida / Cutaneous reactivity to bacterial antigens during the first of life

Se exploró la reactividad cutánea a 100 niños, desde su nacimiento hasta el año de edad, en 3 momentos, recién nacidos, a los 6 meses y al año de edad, con antígenos de staphylococcus albus y Escherichia coli. Los antígenos fueron introducidos por inyección intradémica y la lectura de los resultados se realizó a los 20 minutos (reacción inmediata), 6 horas (reacción rápida) y 48 horas (reacción retardada). Se obtuvo respuestas correlativas al desarrollo del sistema inmunitario en el niño y la historia familiar de enfermedad alérgicas. En este trabajo se pone de manifiesto el desarrollo de la llamada alergia bacteriana y abre una puerta de carácter científico para el uso de las vacunas bacterianas

Asma y Herencia / Asthma and inheritance

Se plantea que el papel de la herencia en la etiología del asma bronquial ha sido objeto de estudio desde hace muchos años. Se realiza esta investigación en niños cubanos con antecedentes familiares de alergia y se analizan la edad en que comenzaron a presentar síntomas de asma bronquial, así como la herencia y otros factores desfavorables que ejercen influencia en el desarrollo de esta enfermedad

Rehabilitación del paciente asmático con deformidad torácica / Rehabilitation of the asthmatic with thoracic deformity

Se realizó un estudio en pacientes asmáticos grado III que presentaban deformidades torácicas como complicación, y en el cual se les indicaron pruebas funcionales respiratorias, medidas antropométricas y resistencia pulmonar antes de un tratamiento fisioterapéutico y 2 años después, dirigido a fortalecer la musculatura respiratoria, el ritmo respiratorio y corregir las deformidades torácicas y posturales. Se expresa que transcurridos 2 años se encontró que, en el 90 % de los pacientes, estas deformidades habían mejorado significativamente; se señala que estos niños tenían como único tratamiento medicamentoso el intal. Se apreció, en el 100 % de los infantes, una mejoría clínica evidente de sus síntomas respiratorios, una disminución significativa de la resistencia de las vías aéreas y un aumento marcado de la capacidad vital. Se concluye que la fisioterapia y los ejercicios respiratorios son de utilidad en el tratamiento a pacientes asmáticos con deformidades torácicas

Reactividad cutánea a antígenos bacterianos durante el primer año de vida / Cutaneous reactivity to bacterial antigens during the first of life

Se exploró la reactividad cutánea a 100 niños, desde su nacimiento hasta el año de edad, en 3 momentos, recién nacidos, a los 6 meses y al año de edad, con antígenos de staphylococcus albus y Escherichia coli. Los antígenos fueron introducidos por inyección intradémica y la lectura de los resultados se realizó a los 20 minutos (reacción inmediata), 6 horas (reacción rápida) y 48 horas (reacción retardada). Se obtuvo respuestas correlativas al desarrollo del sistema inmunitario en el niño y la historia familiar de enfermedad alérgicas. En este trabajo se pone de manifiesto el desarrollo de la llamada alergia bacteriana y abre una puerta de carácter científico para el uso de las vacunas bacterianas