The FGFR1CHMW FGF-2 interaction induces generation of fast and slow mobile FGFR1 molecules. whereas the fast population increased. The transcriptional activation appears to involve conversion of the immobile matrix-bound and the fast nuclear FGFR1 into a slow chromatin-binding population through FGFR1’s interaction with CBP, RSK1, and the high-molecular-weight form of FGF-2. Our findings support a general mechanism in which gene activation is governed by protein movement and collisions with other proteins and nuclear structures. INTRODUCTION A novel gene regulatory mechanism, integrative nuclear FGF receptor-1 signaling (INFS), has been shown to control cell development (Stachowiak 2007 ). In INFS, activation of cell surface neurotransmitter, hormonal or growth factor receptors, and their intracellular messengers (i.e., cAMP) stimulates the release of FGF receptor-1 (FGFR1) from pre-Golgi membranes into the cytosol. The receptor and its ligand, FGF-2, are cotransported into the nucleus by a mechanism that involves importin |gb and engage in the regulation of genes at different chromosomal loci (Reilly and Maher, 2001 ; Stachowiak 2003b ). PTC299 The INFS signaling mechanism involves FGFR1 feeding forward these signals to CREB-binding protein (CBP), a common and essential transcriptional coactivator that acts as a gene activation gating factor (Myers 2003 ; Fang 2005 ; Stachowiak 2007 ). Nuclear FGFR1 executes the release of CBP from its inactive complex with RSK (Fang 2005 ), a process shown to up-regulate gene activities associated with cell differentiation (Nakajima 1996 ). Through the coupled PTC299 activation of CBP by INFS and transcription factors by specific signaling pathways, this signaling mechanism may enable coordinated gene activation by developmental cues and has been referred to as a feed-forward-and-gate signaling (Fang 2005 ; Stachowiak2007 ). Coextraction of FGFR1 with the nucleoplasm, chromatin-associated factors, and the nuclear matrix (NM) as well as its association with active RNA transcription sites and gene promoters indicated both a global and direct role for FGFR1 in gene regulation. Steady-state biochemical analyses suggested that stimulation of gene activities by nuclear FGFR1 occurs in cooperation with CBP as well as nuclear high-molecular-weight (HMW; 23 kDa) FGF-2 and RSK1 and may involve FGFR1 interaction with these proteins (Fang 2005 ; Stachowiak2007 ). In recent years a dynamic picture of transcription regulation has been emerging based on the findings generated from fluorescence recovery after photobleaching (FRAP; Phair and Misteli, 2000 ; Phair 2004 ). The assembly of transcriptional subunits into effective complexes appears to be a dynamic process involving the random collision of the factors involved in transcription (McNally 2000 ; Karpova 2004 ; Stavreva 2004 ). In our previous report we used FRAP to analyze FGFR1 movement and cytoplasmic processing in live cells. There are three distinct pools of the receptor: an immobile pool associated with ER-Golgi vesicles, a slow-moving population (t1/2 = 69 s) associated with cellular membranes, and a fast-moving (t1/2 = 0.2 s) cytosolic pool not associated with membranes (Dunham-Ems 2006 ). The latter of these likely represents the pool of FGFR1 able to enter cell nucleus (Myers 2003 ). The present study used a combination of biophotonics and cell biology tools to further elucidate the mechanisms of gene regulation by the INFS. These tools have provided novel insights into the dynamic nature and molecular proximity of developmental gene regulation. Transcriptional activation by nuclear FGFR1 involves conversion of the immobile NM-bound population and the rapidly diffusing nucleoplasmic population of FGFR1 into a slow chromatin-binding population. FGFR1 transcriptional function is regulated via dynamic associations with the nuclear architecture that is controlled by interactions with CBP, FGF-2, and RSK1. MATERIALS AND METHODS Plasmids pcDNA 3.1, pcDNA3.1-FGFR1, pEGFP-N2, pFGFR1-EGFP, pCMV-RSK1-flag, pRc/RSV-mCBP, and pBI-G expressing anti-sense CBP RNA were described in Fang (2005) . Plasmids expressing HMW (23 kDa) and low-molecular-weight (LMW; 18 kDa) FGF-2 were described in Claus (2003) . HMW-FGF-2-EYFP, HMW-FGF-2-ECFP, LMW-FGF-2-EYFP, and LMW-FGF-2-ECFP were generated by subcloning with NheI/EcoRI (HMW) and EcoRI/HindIII (LMW), respectively, from the previously described vectors pEGFP-23 and pEGFP-18 (Claus 2003 ) into pEYFP and pECFP. FGFR1-ECFP (-EYFP) were constructed by removing enhanced green fluorescent protein (EGFP) from the FGFR1-EGFP construct and cloning the enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), respectively. Antibodies FGFR1 (C-terminal), actin, and tubulin, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The N-terminal FGFR1 mcAb6 was described in (Hanneken 1995 ). FGFR1 rabbit polyclonal.2006;17:2223C2235. increased. The transcriptional activation appears to involve conversion of the immobile matrix-bound and the fast nuclear FGFR1 into a slow chromatin-binding population through FGFR1’s interaction with CBP, RSK1, and the high-molecular-weight form of FGF-2. Our findings support a general mechanism in which gene activation is governed by protein movement and collisions with other proteins and nuclear structures. INTRODUCTION A novel gene regulatory mechanism, integrative nuclear FGF receptor-1 signaling (INFS), has been shown to control cell development (Stachowiak 2007 ). In INFS, activation of cell surface neurotransmitter, hormonal or growth factor receptors, and their intracellular messengers (i.e., cAMP) stimulates the release of FGF receptor-1 (FGFR1) from pre-Golgi membranes into the cytosol. The receptor and its ligand, FGF-2, are cotransported into the nucleus by a mechanism that involves importin |gb and engage in the regulation of genes at different chromosomal loci (Reilly and Maher, 2001 ; Stachowiak 2003b ). The INFS signaling mechanism involves FGFR1 feeding forward these signals to CREB-binding protein (CBP), a common and essential transcriptional coactivator that acts PTC299 as a gene activation gating factor (Myers 2003 ; Fang 2005 ; Stachowiak 2007 ). Nuclear FGFR1 executes the release of CBP from its inactive complex with RSK (Fang 2005 ), a process shown to up-regulate gene activities associated with cell differentiation (Nakajima 1996 ). Through the pHZ-1 coupled activation of CBP by INFS and transcription factors by specific signaling pathways, this signaling mechanism may enable coordinated gene activation by developmental cues and has been referred to as a feed-forward-and-gate signaling (Fang 2005 ; Stachowiak2007 ). Coextraction of FGFR1 with the nucleoplasm, chromatin-associated factors, and the nuclear matrix (NM) as well as its association with active RNA transcription sites and gene promoters indicated both a global and direct role for FGFR1 in gene regulation. Steady-state biochemical analyses suggested that stimulation of gene activities by nuclear FGFR1 occurs in cooperation with CBP as well as nuclear high-molecular-weight (HMW; 23 kDa) FGF-2 and RSK1 and may involve FGFR1 interaction with these proteins (Fang 2005 ; Stachowiak2007 ). In recent years a dynamic picture of transcription regulation has been emerging based on the findings generated from fluorescence recovery after photobleaching (FRAP; Phair and Misteli, 2000 ; Phair 2004 ). The assembly of transcriptional subunits into effective complexes appears to be PTC299 a dynamic process involving the random collision of the factors involved in transcription (McNally 2000 ; Karpova 2004 ; Stavreva 2004 ). In our previous report we used FRAP to analyze FGFR1 movement and cytoplasmic processing in live cells. There are three distinct pools of the receptor: an immobile pool associated with ER-Golgi vesicles, a slow-moving population (t1/2 = 69 s) associated with cellular membranes, and a fast-moving (t1/2 = 0.2 s) cytosolic pool not associated with membranes (Dunham-Ems 2006 ). The latter of these likely represents the pool of FGFR1 able to enter cell nucleus (Myers 2003 ). The present study used a combination of biophotonics and cell biology tools to further elucidate the mechanisms of gene rules from the INFS. These tools have offered novel insights into the dynamic nature and molecular proximity of developmental gene rules. Transcriptional activation by nuclear FGFR1 entails conversion of the immobile NM-bound populace and the rapidly diffusing nucleoplasmic populace of FGFR1 into a sluggish chromatin-binding populace. FGFR1 transcriptional function is definitely regulated via dynamic associations with the nuclear architecture that is controlled by relationships with CBP, FGF-2, and RSK1. MATERIALS AND METHODS Plasmids pcDNA 3.1, pcDNA3.1-FGFR1, pEGFP-N2, pFGFR1-EGFP, pCMV-RSK1-flag, pRc/RSV-mCBP, and pBI-G expressing anti-sense CBP RNA were described in Fang (2005) . Plasmids expressing HMW (23 kDa) and low-molecular-weight (LMW; 18 kDa) FGF-2 were explained in Claus (2003) . HMW-FGF-2-EYFP, HMW-FGF-2-ECFP, LMW-FGF-2-EYFP, and LMW-FGF-2-ECFP were generated by subcloning with NheI/EcoRI (HMW) and EcoRI/HindIII (LMW), respectively,.