Analysis of Site‐Specific Phosphorylation of PTEN by Using Enzyme‐Catalyzed Expressed Protein Ligation

Abstract

The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C terminus. Prior enzymatic characterization of the four monophosphorylated (1p) PTENs by using classical expressed protein ligation (EPL) was complicated by the inclusion of a non‐native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here, we apply subtiligase‐mediated EPL to create wt 1p‐PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with the role of Tyr379 in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p‐PTENs, which are poorly recognized by an anti‐phospho‐PTEN antibody, three of the four wt 1p‐PTENs are recognized by a commonly used anti‐phospho‐PTEN antibody.

Chemoenzymatic Semisynthesis of Proteins

“…471 Critically, the authors used this approach to generate semisynthetic PTEN in a traceless manner through the ligase-catalyzed condensation between a recombinant fragment containing a C-terminal tyrosine α-thioester and a peptide bearing an N-terminal RY dipeptide sequence. 227,471 Semisynthesis of PTEN using EPL previously required the installation of a non-native Cys residue for the ligation reaction, a mutation which has been found to affect the behavior of the protein in cells. 471,484 Using the enzyme-templated approach, Cole and co-workers generated both monophosphorylated 227 and tetraphosphorylated 471 forms of PTEN, which is autoinhibited by these PTMs (see section 7.5.3 for further details).…”

“…227,471 Semisynthesis of PTEN using EPL previously required the installation of a non-native Cys residue for the ligation reaction, a mutation which has been found to affect the behavior of the protein in cells. 471,484 Using the enzyme-templated approach, Cole and co-workers generated both monophosphorylated 227 and tetraphosphorylated 471 forms of PTEN, which is autoinhibited by these PTMs (see section 7.5.3 for further details).…”

Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL

“…In the present work, phospho-PTEN levels remained constant after the addition of FCS (0–90 min), ruling out CT-phosphorylation as the mechanism behind PTEN inactivation after the addition of GF. The present work does not, however, exclude a potential reduction in the phosphorylation of individual CT-Ser and Thr residues since the Ab used for the analysis does not distinguish fully phosphorylated PTEN from single residue phosphorylation defects [ 42 ].…”

Crosstalk between circRNAs and the PI3K/AKT signaling pathway in cancer progression

“…PTEN is a widely mutated tumor suppressor gene that inhibits the oncogenic PI3K/AKT pathway. 113 – 115 PTEN antagonizes the PI3K/Akt pathway by dephosphorylating PIP3 to PIP2, 116 , 117 then induces changes in a variety of cellular biological functions. 118 , 119 Carboxyl-terminal modulator protein (CTMP) could block the transmission of downstream signaling pathways by inhibiting AKT phosphorylation.…”