Project Grant R01GM155497

PP2A/B55ALPHA SUBSTRATE SPECIFICITY AND FUNCTION IN CELL CYCLE PROGRESSION - THE GOAL OF THIS PROPOSAL IS TO FULLY DELINEATE THE MOLECULAR MECHANISMS BY WHICH THE PROMINENT SERINE/THREONINE PROTEIN PHOSPHATASE 2 (PP2A) HOLOENZYME, SPECIFICALLY PP2A/B55A, RECOGNIZES ITS SUBSTRATES THROUGH A NEW HELICAL MOTIF AND TO USE THIS INFORMATION TO IDENTIFY DIRECT PP2A/B55A SUBSTRATES WITH FUNCTIONS IN MITOGENIC SIGNALING AND EARLY CELL CYCLE PROGRESSION. PROTEIN PHOSPHORYLATION IS AN ESSENTIAL REGULATORY MECHANISM IN CELL SIGNALING AND THE REGULATION OF THE CELL CYCLE AND IT IS ALTERED IN CANCER, ALZHEIMER, AND OTHER DISEASES. A LARGE FRACTION OF DEPHOSPHORYLATION IS MEDIATED BY DISTINCT PP2A TRIMERIC HOLOENZYMES. THESE CONSIST OF A SCAFFOLD PROTEIN THAT CONNECTS THE CATALYTIC SUBUNIT TO THE VARIABLE REGULATORY SUBUNIT, WHICH MEDIATES SUBSTRATE SPECIFICITY. THE MOST ABUNDANT HOLOENZYME IS PP2A/B55A. ITS ACTIVITY HAS BEEN IMPLICATED IN MITOGENIC SIGNALING AND EARLY CELL CYCLE PROGRESSION, BUT DETAILS REMAIN UNCLEAR. INITIALLY, IT WAS BELIEVED THAT PP2A/B55A RECRUITED SUBSTRATES THROUGH CHARGE-CHARGE INTERACTIONS BETWEEN THE SURFACE OF B55A AND SUBSTRATES. HOWEVER, OUR GROUNDBREAKING DISCOVERIES CHALLENGED THIS NOTION. WE IDENTIFIED A CONSERVED AMINO ACID MOTIF IN VARIOUS PROTEINS, INCLUDING THE P107 SUBSTRATE AND THE FAM122A INHIBITOR, THAT FOLDS AS AN A-HELIX. THIS HELICAL MOTIF IS ESSENTIAL FOR P107 DEPHOSPHORYLATION AND FOR FAM122A BINDING VIA SIMILAR INTERACTIONS WITH RESIDUES IN A GROOVE ON THE TOP OF B55A. THIS MECHANISM IS DISTINCT FROM THAT USED BY OTHER SER/THR PHOSPHATASES THAT BIND SHORT LINEAR MOTIFS IN INTRINSICALLY DISORDERED PROTEIN REGIONS. GAP: DESPITE THIS ADVANCE, WE REMAIN UNABLE TO IDENTIFY OTHER PP2A/B55A SUBSTRATES USING THIS SHORT HELICAL SEQUENCE. THIS IS DUE TO THE LACK OF A COMPREHENSIVE ASSESSMENT OF AMINO ACID VARIABILITY WITHIN THE HELICAL MOTIF SEQUENCE AND INCOMPLETE DETAIL OF THE MOTIF'S STRUCTURAL PLASTICITY. AS A RESULT, IT REMAINS EXTREMELY DIFFICULT TO IDENTIFY NEW SUBSTRATES, TO DISTINGUISH CORRECTLY VS INCORRECTLY ASSIGNED SUBSTRATES, AND TO DEMONSTRATE THE VERACITY OF CORRECTLY ASSIGNED SUBSTRATES. WE HYPOTHESIZE THAT A HELICAL REGION WITH A DEFINED LENGTH AND A VARIABLE CONSERVED AMINO ACID SEQUENCE IN PP2A/B55A SUBSTRATES MEDIATES BINDING TO THE TOP GROOVE OF B55A VIA SPECIFIC ELECTROSTATIC AND HYDROPHOBIC INTERACTIONS. THIS IS A PRIMARY MECHANISM FOR SUBSTRATE RECOGNITION THAT MODULATES SIGNALING THROUGH THE CELL CYCLE. THESE WILL BE TESTED WITH TWO AIMS: (1) TO DEFINE PP2A/B55A SUBSTRATE SPECIFICITY VIA A SHORT HELICAL MOTIF WITH DEFINED SEQUENCES. (2) TO DISSECT THE PP2A/B55A SUBSTRATE INTERPLAY IN MITOGENIC SIGNALING LINKED TO THE CELL CYCLE RESTRICTION POINT (RP). DEIA AIMS: DIVERSITY AMONG TEMPLE UNIVERSITY UNDERGRADUATES IS EXCELLENT, BUT THE TRANSITION OF UNDERREPRESENTED MINORITY (URM) STUDENTS TO BIOMEDICAL SCIENCE RESEARCH CAREERS OR GRADUATE SCHOOLS IS STILL SEVERELY LIMITED. OUR GOAL IS TO IMPLEMENT A PROGRAM WE HAVE BEEN PILOTING THAT IS GEARED TOWARD ELIMINATING BARRIERS TO JOINING THE SCIENTIFIC RESEARCH WORKFORCE FACED BY URM UNDERGRADUATES AT TEMPLE AND NATIONWIDE. THE PROGRAM WILL RECRUIT 15 STUDENTS WHO WILL WORK IN MENTORS' LABS ON A SPECIFIC PROJECT AND RECEIVE PROGRAMMATIC TRAINING TO PREPARE THEM FOR ADMISSION AND SUCCESS IN BIOMEDICAL RESEARCH PHD PROGRAMS. AIM 1 IS TO DEVELOP RESEARCH SKILLS AND KNOWLEDGE AND AIM 2 IS TO DEVELOP CAREER SKILLS BY PROVIDING INSTRUCTION IN FELLOWSHIP WRITING, GRADUATE SCHOOL APPLICATION, ORAL AND WRITING SCIENTIFIC COMMUNICATION, AND SCIENTIFIC NETWORKING. SUCCESSFUL COMPLETION OF THE SCIENTIFIC AIMS WILL FACILITATE IDENTIFICATION OF PP2A/B55A SUBSTRATES AND THEIR VALIDATION IN MULTIPLE PATHWAYS AND CELLULAR PROCESSES OFTEN DEREGULATED IN DISEASE PROVIDING NOVEL TARGETS FOR THERAPEUTIC INTERVENTION. THE DEIA TRAINING PROGRAM WILL GUIDE URM STUDENTS TO PROFESSIONAL RESEARCH CAREERS.