Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc

Abstract

Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility. Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility. Cell motility is controlled by a complex network of signals that are initiated by binding to the extracellular matrix. Understanding the biochemical mechanisms that control cell migration is necessary for better comprehension of processes like wound healing, embryonic development, and angiogenesis as well as cancer metastasis (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3226) Google Scholar). PKA 3The abbreviations used are: PKA, protein kinase A; AKAP, A-kinase anchoring proteins; AKAR1, A-kinase activity reporter; FRET, fluorescence resonance energy transfer; Fc, sensitized acceptor emission or corrected FRET; PDE, phosphodiesterase; PKI, protein kinase inhibitor; RII, type II regulatory; NT, non-targeting; LPA, lysophosphatidic acid; PIPES, 1,4-piperazinediethane-sulfonic acid; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interfering RNA; IBMX, isobutylmethylxanthine.3The abbreviations used are: PKA, protein kinase A; AKAP, A-kinase anchoring proteins; AKAR1, A-kinase activity reporter; FRET, fluorescence resonance energy transfer; Fc, sensitized acceptor emission or corrected FRET; PDE, phosphodiesterase; PKI, protein kinase inhibitor; RII, type II regulatory; NT, non-targeting; LPA, lysophosphatidic acid; PIPES, 1,4-piperazinediethane-sulfonic acid; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interfering RNA; IBMX, isobutylmethylxanthine. is an important regulator of cell signaling and various biological functions (2Meinkoth J.L. Alberts A.S. Went W. Fantozzi D. Taylor S.S. Hagiwara M. Montminy M. Feramisco J.R. Mol. Cell. Biochem. 1993; 127-8: 179-186Crossref Scopus (123) Google Scholar, 3Taylor S. Knighton D.R. Zheng J. TenEyck L.F. Sowadski J.M. Faraday Discuss. 1992; 93: 143-153Crossref PubMed Google Scholar, 4Iyengar R. Science. 1996; 271: 461-463Crossref PubMed Scopus (120) Google Scholar). Previous studies have shown that cell motility is delicately controlled by synthesis and breakdown of cAMP through its effects on PKA. PKA regulates key signaling events that are critical for actin cytoskeletal remodeling and cell polarization during migration, including control of the activation states of RhoA, Rac, cdc42, Pak, and c-Abl. For example, PKA is known to inhibit the activation of RhoA, whereas it is required for the activation of Rac1, two proteins that are spatially regulated during cell migration. Therefore, it has been suggested that PKA activity in migrating cells is spatially regulated (5O'Connor K.L. Shaw L.M. Mercurio A.M. J. Cell Biol. 1998; 143: 1749-1760Crossref PubMed Scopus (136) Google Scholar, 6O'Connor K.L. Mercurio A.M. J. Biol. Chem. 2001; 276: 47895-47900Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Howe A.K. Baldor L.C. Hogan B.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14320-14325Crossref PubMed Scopus (69) Google Scholar, 8Howe A.K. PubMed Scopus Google Scholar, J. A.K. J. Cell Biol. PubMed Scopus Google Scholar). The for the formation of gradients and cell motility is that PKA activity gradients the of these gradients in cells is to the of gradients and the mechanisms are The of cAMP was suggested J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Google and has been shown to the control of its J. R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, W. Mol. Cell Biol. PubMed Scopus Google Scholar, M. Cell Biol. PubMed Scopus Google Scholar). control of cAMP is by the of and of cAMP PKA activity are to in a This is on the that cAMP is generated by and by that the two of cAMP are spatially of PKA activity as a of the anchoring of PKA and to A-kinase anchoring proteins has been in a of cell K.L. S. R. J. 2001; PubMed Scopus Google Scholar, M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The anchoring of PKA through the binding of the type II to the of activity and cAMP generated the activity of PKA. PKA in to cAMP synthesis and is to control signaling of PKA W. Mol. Cell Biol. PubMed Scopus Google Scholar, M. Cell Biol. PubMed Scopus Google Scholar). we have the and to the that activity gradients We and (5O'Connor K.L. Shaw L.M. Mercurio A.M. J. Cell Biol. 1998; 143: 1749-1760Crossref PubMed Scopus (136) Google Scholar, 6O'Connor K.L. Mercurio A.M. J. Biol. Chem. 2001; 276: 47895-47900Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Howe A.K. Baldor L.C. Hogan B.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14320-14325Crossref PubMed Scopus (69) Google Scholar, 8Howe A.K. PubMed Scopus Google have that polarization and migration of cells are on cAMP synthesis and Here, we to the existence of gradients in migrating cells using the fluorescence resonance energy PKA A-kinase activity reporter and signaling that PKA including cAMP and PKA the formation of these gradients. Cell and A carcinoma cells to in with AKAR1, a non-phosphorylatable mutant of AKAR1 of of J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google or using cells by and with for with IBMX, or protein kinase or for with or control for as with laminin-1 in the of for at for at with PIPES, and with with protein to fluorescence in for For using cells to by and in of for stimulated with lysophosphatidic for or using a with a of The using two of and and two of The of fluorescence of cyan fluorescent protein at was using a The of fluorescence of yellow fluorescent protein acceptor of at and the fluorescence of at using a using with a of a of of and a of and an of of the cell to cell of AKAR1 the and the as to the of for however, the the of the For the used for cells AKAR1 YFP, or into for was on the of and a was using a to the J. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). a corrected we the to for signals in the as due to the and YFP, by the J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Scholar, J. S. J. Full Text PDF PubMed Scopus Google a and The by the acceptor emission acceptor the as by J. M. J. Full Text Full Text PDF PubMed Scopus Google the of the used for signals in for of the reporter the and a of J. Full Text Full Text PDF PubMed Scopus Google Scholar). The and using cells or a was the of for whereas the was the of for For of a and at using at two cells and of that was no the at the that the the for the was The used to sensitized emission of the for on the The to the of using acceptor emission and as a as J. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). For cell the of in was that the the edge of the the For the of and in a siRNA, and The the to of the gradients of are in cells. This was used to the the leading edge to the cell and to For data cells shown cells at two and a of PKA PKA was by of cell as K.L. Biochem. PubMed Scopus Google Scholar). cells on in the of and in a and A of was as a and the was with to protein for at and PKA and for the PKA and by of using a to that signals in the cells with of AKAR1 or in with specific for or a control as M. K.L. 2005; PubMed Scopus Google Scholar). for are and in for and for using or cell migration. using and protein was on a and to The was with and for by or the of with in to the and at for the with a and cells on the surface with and as K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar). for at with PIPES, and in for with in and in in for and with of phospho-PKA substrate Cell in at cells with or with and by of the and using the and and was as necessary of the the of PKA activity in migrating we the AKAR1 J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). AKAR1 a that be by PKA and the binding of to are by and that binding of the to the PKA is The by is initiated by PKA and is by an the of for a type of PKA a cell for the of PKA activity gradients during migration, we the A migration A cells to and migrate randomly on laminin-1 by signaling through the and Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar). A cells form a polarized in the leading edge is with a of these cells that cells migrate in a the are and the cell in the of the not Therefore, we are to migrating cells by the of a the leading edge and at the the of A cells with AKAR1, a non-phosphorylatable mutant of AKAR1, or to migrate on laminin-1 and of cells with AKAR1 and cells that cells the AKAR1 no in when with cells for data not of for of fluorescent in the using a with a for the of sensitized acceptor emission or corrected J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. as for cells with or This of has been to J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google that are of the of into that during acceptor M. 2005; PubMed Scopus Google Scholar). The was to the of and as a with acceptor as suggested J. M. J. Full Text Full Text PDF PubMed Scopus Google a for PKA the fluorescence the cell that of cell and Therefore, was necessary to shown in A cells to migrate on laminin-1 show a with a of at the leading PKA activity in and the of a of PKA activity of of cells of the show that the in the that PKA activity is spatially the cell during migration. that the gradients in are specific to PKA activity and not a of cell or of AKAR1 cells with the mutant of AKAR1 that be by PKA not as J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). on laminin-1 that for to the AKAR1 and in an In we show that cells the mutant and of in the as shown by the of of cells and and we to that the was specific and a of PKA For these we cells with and the and during migration on A cells are in the that to form and in PKA is as for these processes and not K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google PKA activity to K.L. Mercurio A.M. J. Biol. Chem. 2001; 276: 47895-47900Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Howe A.K. Baldor L.C. Hogan B.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14320-14325Crossref PubMed Scopus (69) Google Scholar, M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, the of PKA to the in cells be in Here, we find that cells with H-89 of cells or of of cells show and no PKA activity gradients to cells the AKAR1 the PKA activity gradients are in the in the cell is the the cell that our are not a of in the of AKAR1 or the at the we of cells. and to is in the cell and is the leading was at the leading edge and the as and shown in the in we that an with in cells. in these cells the of a In the cells or or cells show no or a in these data that the detected with AKAR1 in migrating cells is due to the PKA of the AKAR1 and is in with the by the of AKAR1 J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). PKA activity gradients the we of migrating A cells the AKAR1 at in the gradients are the basal data that the PKA activity gradients are with the basal to the are during the that PKA gradients form the as well as the leading we to signaling known to cAMP signaling are in the formation of these PKA activity gradients. and of are to control cAMP gradients in cell and the of signaling D.R. Biochem. J. PubMed Scopus Google Scholar). activity has been shown to be important for cell motility (5O'Connor K.L. Shaw L.M. Mercurio A.M. J. Cell Biol. 1998; 143: 1749-1760Crossref PubMed Scopus (136) Google Scholar, K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar, J. Cell Sci. PubMed Scopus Google Scholar). cAMP A cells with the IBMX, on and to to shown in we find that cells with of cells PKA activity gradients as by the with the of the and of the that the of the PKA is with cells. cells no longer a polarized of PKA that the is not restricted to a or leading edge is the of the cell to This observation is in with a of cell polarization and in cell motility that has been by inhibition (5O'Connor K.L. Shaw L.M. Mercurio A.M. J. Cell Biol. 1998; 143: 1749-1760Crossref PubMed Scopus (136) Google Scholar, K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar, J. Cell Sci. PubMed Scopus Google Scholar). The or the is not a of the be used to in activity are a For we the PKA activity in cells a that was to PKA. This is on the ability of an kinase to a as that in the PKA The of a to in the PKA it the PKA with we a protein the to and to to PKA cell and a of the of kinase the cell The of has been and to be kinase K.L. Biochem. PubMed Scopus Google Scholar). the we find that of the PKA is when cells are with the in cAMP inhibition and PKA activation are on basal cAMP synthesis W. Mol. Cell Biol. PubMed Scopus Google Scholar, M. Cell Biol. PubMed Scopus Google we used with to activation of PKA In cells with and PKA activity gradients of of cells as shown by and by and cell and polarization data that the PKA activity gradients as a of of cAMP synthesis the of the PKA that our data are of PKA we to using phospho-PKA as has been A. J. Mol. Biol. Cell. PubMed Scopus Google Scholar). For these A cells or with IBMX, or on and with phospho-PKA antibodies. shown in H-89 not for the phospho-PKA whereas and are using of the cell to that of the that the with phospho-PKA substrate with that PKA In the cells we find in the it the cell the of the cell is the This is by the in the and cell that the phospho-PKA substrate of the is that of the cell A.K. Baldor L.C. Hogan B.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14320-14325Crossref PubMed Scopus (69) Google that PKA activity and in of show that is PKA anchoring PKA anchoring is in the formation of PKA activity we binding to and PKA and the control in the by two to J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). shown in the control not inhibit cell polarization and the PKA activity gradients for cells in of cells anchoring the formation of cell and the formation of PKA activity gradients of of cells of cells with these not PKA activity as shown in its data that cAMP with PKA anchoring is for the of PKA gradients. we to be in PKA activity of the of these gradients to the basal and the effects of PKA on the actin we our on and actin and we find that and are not in A AKAP-Lbc, and are not is with specific W. Mol. Cell Biol. PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google Scholar, J. S. J. Science. PubMed Scopus Google and is with Cell 2005; PubMed Scopus Google we our on the actin is a protein functions as a for of lysophosphatidic D. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). the used to PKA anchoring is the binding of J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). For these we used to the of of AKAP-Lbc, we A cells on cells with and as K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar). cells with siRNA, and cells with or and with not A cells with the AKAR1 and or the and with cells with AKAR1 that AKAR1 and AKAR1 cells PKA activity gradients at the leading edge of the cell The a the cell is the cell with in PKA activity the as by the of the when with the of was confirmed by that the in is we the of the using cells in the the for and cells are and whereas for or cells and using the a of not these data that to the formation of PKA activity gradients. we to the of cell migration. For these control and cells to migration on shown in a in migration was with the of data are in with the observation that not the formation of PKA activity gradients for cells migrating on that the existence of PKA activity gradients in cell of migration, we cells stimulated with carcinoma cells migrate M. K.L. J. Cell PubMed Scopus Google Scholar). migration is inhibited by PKA and inhibition using H-89 or and IBMX, not PKA activity gradients in these cells or stimulated with for cells a of phospho-PKA substrate at the leading edge with the cell these data that PKA activity gradients in migrating cells and are to signaling events at the leading edge of migrating cells. Previous studies have suggested that PKA activity or inhibit actin cytoskeletal and cell migration. This of PKA a of for regulation of PKA activity during cell migration. We and K.L. Mercurio A.M. J. Biol. Chem. 2001; 276: 47895-47900Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Howe A.K. Baldor L.C. Hogan B.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14320-14325Crossref PubMed Scopus (69) Google Scholar, 8Howe A.K. PubMed Scopus Google Scholar, J. A.K. J. Cell Biol. PubMed Scopus Google have suggested that PKA activity gradients in migrating cells to polarization of cells and signaling for the of actin cytoskeletal The that PKA activity is in migrating cells A.K. Baldor L.C. Hogan B.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14320-14325Crossref PubMed Scopus (69) Google Scholar). a polarized of PKA and activity to the during migration. This of and PKA activity with an of of the PKA and data the existence of PKA activity gradients in migrating cells and that these gradients are to key of actin cytoskeletal the we find that these gradients are in to with the extracellular matrix. data are in with using a that PKA activity is at the leading edge in cells migrating on a using and A. J. Mol. Biol. Cell. PubMed Scopus Google Scholar). with our we and collagen these a in the formation of PKA activity gradients. extracellular have been in control of cAMP and PKA through the activation of (5O'Connor K.L. Shaw L.M. Mercurio A.M. J. Cell Biol. 1998; 143: 1749-1760Crossref PubMed Scopus (136) Google of cAMP synthesis Cell Biol. PubMed Scopus Google and with PKA anchoring J. Cell Sci. 2001; PubMed Google Scholar). further the PKA activity and are in migrating cells. In our data that PKA anchoring with cAMP synthesis and of cAMP is for the of PKA whereas activity the formation of the to the leading edge migrating cells. the of in cell migration has been specific to cell migration is not well we find that a AKAP, AKAP-Lbc, a in the formation of PKA activity gradients cell migration. Previous studies on have its in regulation D. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, D. A. J. Cell Biol. PubMed Scopus Google and the activation of protein kinase Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The of its in the regulation of PKA of its through the of a D. S. J. PubMed Scopus Google Scholar, J. S. Taylor Taylor A. J.R. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We find that not to as a in A cells in to binding A. M. and the that A cells and during migration K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar). Therefore, we that the of in cell migration in the is through its kinase anchoring its This is with of anchoring protein in the its kinase anchoring activity in the of signaling events that the of J. D. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). We further that to PKA to PKA to cell migration. however, PKA activity gradients in the of the This that to PKA activity during cell migration. and is a major actin J. PubMed Scopus Google that is to a critical in cell migration through actin cytoskeletal as the to the complex and small to the formation of the actin J. Cell Sci. 2001; PubMed Google Scholar). is a as a of cell migration through its functions at the leading edge S. M. J. R. M. Mol. Biol. Cell. PubMed Scopus Google Scholar, J. J. Biochem. Cell Biol. PubMed Scopus Google Scholar). is a as it is on the it is to activity W. Mol. Cell Biol. PubMed Scopus Google Scholar). studies are to be in PKA activity formation and to the events that control cell motility. of our is that activity is not in the formation of PKA activity gradients is critical for these gradients We find that cells that are migrating PKA activity gradients that are restricted to the leading edge of the cell and the are inhibited and basal PKA activity are PKA activity gradients are no longer polarized form due to the of cAMP the PKA is to control key signaling events at the leading the of polarization of PKA gradients a of cell migration. This is by studies on the of during cell polarization and migration (5O'Connor K.L. Shaw L.M. Mercurio A.M. J. Cell Biol. 1998; 143: 1749-1760Crossref PubMed Scopus (136) Google Scholar, K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar, J. Cell Sci. PubMed Scopus Google Scholar). This observation is to the of in formation on J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and that cAMP M. Cell Biol. PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google however, it is by studies that that are to signaling spatially J. J. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). are known to be or to as W. Mol. Cell Biol. PubMed Scopus Google Scholar, D.R. Biochem. J. PubMed Scopus Google Scholar). The of the of the cAMP have been to W. Mol. Cell Biol. PubMed Scopus Google Scholar, K.L. S. R. J. 2001; PubMed Scopus Google Scholar). have been shown to be regulated by as shown with J. D. W. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). as to and the of the at the of cAMP the of cAMP to PKA, to a PKA activity and the polarization to PKA activity and formation further we our our as of PKA activity the AKAR1 reporter the of a is initiated by PKA and by a it is the by PKA that the signaling events of PKA. We and have PKA as a critical kinase during migration through the of K.L. Mercurio A.M. J. Biol. Chem. 2001; 276: 47895-47900Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Howe A.K. Baldor L.C. Hogan B.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14320-14325Crossref PubMed Scopus (69) Google Scholar, 8Howe A.K. PubMed Scopus Google Scholar, J. A.K. J. Cell Biol. PubMed Scopus Google Scholar). is by its during cell migration is not are known to as well as W. Mol. Cell Biol. PubMed Scopus Google Scholar). is an that as for and of the of PKA through its kinase and to PKA signaling be an important for with the of PKA activity in cell migration, we the cell migration, and of at the leading are stimulated Cell Biol. PubMed Scopus Google Scholar). PKA to as as shown in to J. Cell Sci. 2001; PubMed Google and the actin is of its K.L. Mercurio A.M. J. Biol. Chem. 2001; 276: 47895-47900Abstract Full Text Full Text PDF PubMed Scopus (122) Google PKA activation through is restricted to the leading The polarization of PKA activity is controlled through the of and PKA and through the inhibition of PKA controlled through the of cAMP by This of the inhibition and of PKA be to gradients that are required for in and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This PKA activity its effects on of a is RhoA, is by PKA K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar, M. R. M. J. J. 1996; PubMed Scopus Google Scholar, J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In A cells K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google as well as cells of J. Cell Biol. PubMed Scopus Google Scholar, M. Mol. Cell. Biol. Scopus Google Scholar, M. Mol. Cell. Biol. 2005; Google on the of during cell migration K.L. Mercurio A.M. J. Cell Biol. PubMed Scopus Google Scholar, M. Mol. Cell. Biol. 2005; Google it is to be B.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This is that for PKA that PKA and are spatially at the leading to the of of cell migration not of these signaling for PKA regulation at the leading edge cdc42, Rac, and a 8Howe A.K. PubMed Scopus Google Scholar). The and of these proteins for the formation of actin cytoskeletal that is required for cell and of studies have PKA in the activation of the that PKA the polarization of activity at the leading edge to migration A. J. Mol. Biol. Cell. PubMed Scopus Google Scholar, Baldor L.C. A.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a we that PKA activity of to the leading edge the of the a that is required for cell migration (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3226) Google Scholar). In summary, our the existence of PKA activity gradients in migrating cells and that these PKA activity gradients are by the of cAMP synthesis and PKA anchoring through specific as AKAP-Lbc, whereas activity is important for the polarization of these gradients. We and for M. and for for for and and for