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P A Klekotka

Publications and source records attributed to P A Klekotka.

6 recordsLinked to original sources
PubMed2001

Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.

Amino Acid Sequence↗
PubMed2001

Mammary epithelial cell-cycle progression via the alpha(2)beta(1) integrin: unique and synergistic roles of the alpha(2) cytoplasmic domain.

The alpha(2)beta(1) integrin supports cell-cycle progression of mammary epithelial cells adherent to type I collagen matrices. Integrin collagen receptors containing the alpha(2) cytoplasmic domain stimulated expression of cyclin E and cyclin-dependent kinase (cdk)2, resulting in cyclin E/cdk2 activation in the absence of growth factors other than insulin. Integrin collagen receptors in which the alpha(2) cytoplasmic domain was replaced by the alpha(1) cytoplasmic domain or an alpha(2) subunit cytoplasmic domain truncated after the GFFKR sequence failed to stimulate cyclin E/cdk2 activation or entry into S phase in the absence of growth factors. Although overexpression of cyclins D or E or cdk2 in cells expressing the integrin collagen receptor with the alpha(1)-integrin cytoplasmic domain did not restore G(1) progression when mammary epithelial cells adhered to type I collagen, co-expression of cyclin E and cdk2 did rescue the ability of the transfectants to enter S phase. Activation of cyclin E/cdk2 complex by mammary epithelial cells required synergy between adhesion mediated by an integrin collagen receptor containing the alpha(2)-integrin subunit cytoplasmic domain and the insulin receptor.

Breast↗
PubMed2000

alpha 2 integrin subunit cytoplasmic domain-dependent cellular migration requires p38 MAPK.

The alpha(2) integrin subunit cytoplasmic domain uniquely supported epidermal growth factor (EGF)-stimulated migration on type I collagen. p38 MAP kinase- and phosphatidylinositol 3-kinase-specific inhibitors, but not a MEK-specific inhibitor, eliminated EGF-stimulated and unstimulated alpha(2)-cytoplasmic domain-dependent migration. Following adhesion to collagenous matrices, cells expressing the full-length alpha(2) integrin subunit, but not cells expressing a chimeric alpha(2) integrin subunit in which the alpha(2)-cytoplasmic domain was replaced by the cytoplasmic domain of the alpha(1)-subunit, exhibited sustained and robust phosphorylation of p38 MAP kinase. Expression of dominant negative p38 MAP kinase inhibited alpha(2)-cytoplasmic domain-dependent, EGF-stimulated migration as well as unstimulated migration on collagen. Expression of constitutively active Rac1(Val-12) augmented p38 MAP kinase activation and alpha(2)-cytoplasmic domain-dependent migration. It also rescued the ability of cells expressing the alpha(1)-cytoplasmic domain to activate p38 MAPK and to migrate. These results suggest that the alpha(2) integrin cytoplasmic domain uniquely stimulates the p38 MAP kinase pathway that is required for unstimulated and EGF-stimulated migration on type I collagen.

Animals↗
PubMed1995

Inactivation of Escherichia coli-expressed rabbit cytochrome P-450 2C enzymes by 17 beta-substituted steroids.

The specific inactivation of rabbit cytochromes P-450 2C by 17 beta-substituted steroids has been investigated by using purified, Escherichia coli-expressed enzymes. The expressed P-450s provided a means to characterize accurately the effects of 21,21-dichloroprogesterone, 21,21-dichloropregnenolone, 21-chloro-21-fluoropregnenolone, pregn-5,20-diene-3 beta-ol and pregn-4,20-diene-3-one on progesterone hydroxylation by P-450 2C5, 2C4, 2C3 and 2C3v. Previous studies using rabbit liver microsomes had suggested that 21-chloro-21-fluoropregnenolone is a selective inactivator of 2C5, a progesterone 21-hydroxylase. Studies of the expressed P-450 2C forms showed little selectivity of 21,21-dichloroprogesterone, pregn-5,20-diene-3 beta-ol or pregn-4,20-diene-3-one, whereas 21,21-dichloropregnenolone and 21-chloro-21-fluoropregnenolone preferentially inactivate 2C5. The data indicate the importance of progesterone 21-hydroxylase activity in facilitating selective mechanism-based inactivation of 2C subfamily P-450s by 21,21-dihalogenated steroids. Studies of the inactivation of P-450 2C16, a progesterone 16 alpha-hydroxylase, by the three dihalogenated steroids yielded results consistent with previous findings of 16 alpha-hydroxylase inactivation in rabbit liver microsomes from the inbred B/J strain. Additionally, two mutants, 2C3v:V113A and 2C3v:V113A, T364N were created which confer progesterone 21-hydroxylation on 2C3v. The single mutant, a 6 beta- and 21-hydroxylase, is inactivated rapidly by all three of the 21,21-dihalogenated steroids, whereas the double mutant, a 16 alpha- and 21-hydroxylase, is preferentially inactivated by 21,21-dichloroprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Structural determinants of cytochrome P450 2B1 specificity: evidence for five substrate recognition sites.

Twelve site-directed mutants of rat cytochrome P450 2B1 distributed over seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined using androstenedione and testosterone as substrates. Substitutions at positions 303, 360, and 473 did not markedly affect the regio- or stereoselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-4), and 363 and 367 (SRS-5) exhibited markedly different steroid metabolite profiles compared with parental P450 2B1. In particular, the Phe-206-->Leu substitution conferred androgen 6 alpha- and testosterone 7 alpha-hydroxylase activities, and the Thr-302-->Ser substitution suppressed androgen 16 beta-hydroxylation in favor of androstenedione 16 alpha- and testosterone 15 alpha-hydroxylation. Replacement of Val-363 or Val-367 with Ala conferred androgen 15 alpha-hydroxylase and 6 beta-hydroxylase activities, respectively, and suppressed susceptibility to mechanism-based inactivation by the P450 2B1-selective chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367-->Ala mutant was also resistant to chloramphenicol itself. The Leu mutant at position 363 exhibited increased specificity for androstenedione and testosterone 16 beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most interestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face and hydroxylation on the D-ring or the B-ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗